Tal effector means useful for partial or full deletion of dna tandem repeats

ABSTRACT

The application relates to means, which derive from TAL effectors and TALENs. The structure of the means of the application is especially adapted for partial or full deletion of at least one DNA tandem repeat, more particularly for partial or full deletion of at least one DNA tandem repeat in a double-stranded DNA, more particularly for partial or full deletion of at least one DNA tandem repeat, which is contained in a double-stranded DNA and, which forms a complex secondary structure, such as a hairpin, a triple helix or a tetraplex secondary structure. The means of the application are notably useful in the treatment and/or prevention and/or palliation of a disease or disorder involving at least one DNA tandem repeat, such as DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2, SCA10, SPD1, OPMD, CCD, HPE5, HFG syndrome, BPES, EIEE1, FRAXA, FXTAS and FRAXE.

FIELD OF THE INVENTION

The application relates to means, which derive from transcriptionactivator-like (TAL) effectors, more particularly from TAL effectorendonucleases (TALENs).

The means of the application are notably useful for fully or partiallydeleting a DNA tandem repeat, more particularly for fully or partiallydeleting a DNA tandem repeat in a double-stranded DNA molecule, moreparticularly for fully or partially deleting an expanded DNA tandemrepeat in a double-stranded DNA molecule.

The application also relates to medical and biotechnologicalapplications, more particularly in the field of diseases and disordersinvolving expanded DNA tandem repeats in double-stranded DNA molecules,such as trinucleotide repeat diseases or disorders, tetranucleotiderepeat diseases or disorders or pentanucleotide repeat diseases ordisorders.

BACKGROUND OF THE INVENTION

DNA tandem repeats occur frequently in double-stranded DNAs ofeukaryotic genomes, more particularly of the human genome. DNA tandemrepeat units of 2, 3, 4, 5 or even more nucleotides can be observed in agenome at different frequencies and locations (exons, introns,intergenic regions). DNA tandem repeats are prone to recombinationand/or random integration events, and are considered to be at the centerof species evolution.

However, expansion in the length of a DNA tandem repeat can result indeleterious effects on gene function, leading to disease or disorder.Expansion in DNA tandem repeat is known to underlie about 20 severeneurological and/or muscular and/or skeletal diseases or disorders(McMurray 2010).

Over the last 20 years or so, it was demonstrated that replicationslippage, double-strand break repair, base excision repair, nucleotideexcision repair, basically any mechanism involving de novo DNA synthesiswithin a DNA tandem repeat, are involved in DNA tandem repeat expansion.However, the precise mechanisms are still obscure.

A large amount of studies were devoted to understanding the mechanismsresponsible for large trinucleotide repeat expansions, using modelsystems as diverse as bacteria, yeast, drosophila, mice or human celllines.

Richard et al. 1999 and Richard et al. 2003 demonstrated that theinsertion of a recognition site for the rare cutter endonuclease I-SceIor HO between two short (CAG)_(n) repeats leads to the induction of adouble-strand break (DSB) by said endonuclease, resulting incontractions or expansions of the repeat domain. However, the efficacyof such engineered nucleases is highly variable depending on the genomictarget tested, and requires the insertion of the endonucleaserecognition site.

Zinc-finger nucleases (ZFN) were developed for targeted gene editing ineukaryotes. They were built by fusing modular zinc-finger DNA-bindingdomains to the catalytic domain of the Fok I endonuclease (Mittelman etal. 2009). However, they induce high toxicity and a high frequency ofoff-target mutations, probably due to recognition and cutting of manydegenerate sequences differing only slightly from the targeted sequence.

Hence, the available prior art means are not fully adapted to thedeletion of an (expanded) DNA tandem repeat in a double-stranded DNA,and are not adapted to medical applications.

Furthermore, an expanded DNA tandem repeat domain in a double-strandedDNA, such as those observed in pathological conditions, poses particulartechnical problems. Indeed, such an expanded DNA tandem repeat domainforms a complex secondary structure, such as a hairpin, a triple helixor a tetraplex secondary structure, which hinders or complicatesaccessibility to appropriate cleavage and which may promote repeatexpansion during DSB repair (Richard et al. 2000).

Appropriate means should allow size reduction of the (expanded) DNAtandem repeat down to a non-pathological level, even when said(expanded) DNA tandem repeat has a complex secondary structure, such asa hairpin, a triple helix or a tetraplex secondary structure.

Appropriate means should also be as less toxic as possible to allowsurvival of the cell, and induce as less side mutations or alterationsas possible. Advantageously, they should be sufficiently specific toavoid off-targets cleavage as much as possible.

The application provides means, which can achieve these goals.

SUMMARY OF THE INVENTION

The means of the application derive from TAL effectors and TALENs. Thestructure of the means of the application is especially adapted forpartial or full deletion of at least one DNA tandem repeat, moreparticularly for partial or full deletion of at least one DNA tandemrepeat in a double-stranded DNA, more particularly for partial or fulldeletion of at least one DNA tandem repeat, which is contained in adouble-stranded DNA and, which forms a non-linear secondary structure,such as a hairpin, a triple helix or a tetraplex secondary structure.The means of the application are especially adapted for partial or fulldeletion of at least one (expanded) DNA tandem repeat in adouble-stranded DNA, such as those observed in pathological conditions.

The application relates to the subject-matter as defined in the claimsas filed and as herein described.

More particularly, the application relates to DNA-binding polypeptidesand to products deriving therefrom such as nucleic acids, vectors,cells, liposomes, nanoparticles, sets, compositions, kits,pharmaceutical compositions, medicaments and drugs.

The application also relates to uses of said products and to methodsinvolving at least one of said products, more particularly in themedical field.

The products of the application are notably useful in the treatmentand/or prevention and/or palliation of a disease or disorder involvingat least one DNA tandem repeat, more particularly of a trinucleotide,tetranucleotide or pentanucleotide disease or disorder, such as DM1,SCA8, SCA12, HDL2, SBMA, HD, DRPLA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17,PSACH, DM2, SCA10, SPD1, OPMD, CCD, HPE5, HFG syndrome, BPES, EIEE1,FRAXA, FXTAS and FRAXE (cf. Tables 6, 7 and 8 below).

The means of the application allow size reduction of the DNA tandemrepeat down to a non-pathological level at a high efficacy rate (near100% in heterozygous and homozygous yeast cells).

No increase in the mutation rate was detected. No large genomicrearrangement, such as aneuploidy, segmental duplication ortranslocation, was detected.

According to an advantageous aspect of the application, the means of theapplication do not induce any length alteration or mutation atoff-target locations, e.g., in non-pathological genes, which comprisethe same repeat unit as the pathological gene.

It is believed that it is the first demonstration of the induction of ashortening of a DNA tandem repeat in a double-stranded DNA to lengthsbelow pathological thresholds in humans, with 100% efficacy and a highspecificity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: schematic representation of the experiments of example1.

FIG. 1A: Plasmids pCLS9996 (C.N.C.M. deposit number I-4804; C.N.C.M.deposit date: 10 Oct. 2013) and pCLS16715 (C.N.C.M. deposit numberI-4805; C.N.C.M. deposit date: 10 Oct. 2013), carrying the two TALENarms were respectively transformed into GFY40 strain or GFY6162-3D.Haploids were crossed and diploids containing both TALEN arms wereselected on SC-Leu supplemented with G418 sulfate. As a control, thesplit-TALEN left arm carried by pCLS9984 was transformed in GFY6162-3D,crossed to GFY40 carrying the TALEN right arm, and diploids wereselected as before.

FIG. 1B: Sequences recognized by both TALE DNA-binding domains and bythe split-TALE. The length of the spacer, which is appropriate to inducea DSB was deduced from repeat tract lengths analyzed in surviving cellsafter TALEN induction (length of 18 bp).

FIGS. 2A to 2D: Molecular analysis of survivors after TALEN induction.

FIG. 2A: Survival after galactose induction (ratio of CFU on galactoseplates over CFU on glucose plates, after 3-5 days of growth at 30° C.).

FIG. 2B: Molecular analysis of heterozygous diploids(SUP4-opal/sup4-(CAG)).

FIG. 2C: PCR amplification of DNA extracted from survivors. When bothalleles are present, bands of slightly different sizes corresponding touncut alleles are visible in both lanes (arrow labeled “Uncut”), alongwith restriction products of cut alleles (arrows labeled “Cut”). Whenonly the SUP4-opal allele is present, no cut product is detected in the‘I’ lane (clones 8 and 11 to 20).

FIG. 2D: Molecular analysis of homozygous diploids(sup4-(CAG)/sup4-(CAG)). Same as FIG. 2B, except that total genomic DNAwas digested with Ssp I.

FIGS. 3A to 3D: Karyotypes and sequencing of TALEN-induced yeastcolonies.

FIG. 3A: Sanger sequencing of survivors.

FIG. 3B: Two models proposing how heterozygous and homozygous repeatsmay be formed following TALEN induction.

FIG. 3C: Deep sequencing of yeast genomes from yeast colonies isolatedon glucose or galactose plates.

FIG. 3D: Pulse-field gel electrophoresis of red and white colonies aftergalactose induction.

FIG. 4: Southern blots (left: strains GFY6161-3C (MATa leu2Δ1 his3Δ200lys2Δ202 ade2-opal sup4::(CAG)₃₀) and GFY6162-3D; right: transformantsGFY6162-3C/1 and GFY6162-3D/2).

DETAILED DESCRIPTION OF THE INVENTION

The application relates to the subject-matter as defined in the claimsas filed and as herein described.

The means of the application derive from means, which were created forgenome editing, i.e., Transcription Activator-Like (TAL) effectors andTAL effector endonucleases (TALENs).

TAL effectors and TALENs have been described e.g., in Boch et al. 2009,Moscou et al. 2009, Bogdanove and Voytas 2011, Cermark et al. 2011,Bedell et al. 2012, Beurdeley et al. 2012, WO 2011/072246 (and itsnational counterparts, more particularly its US counterpart(s)(including the US continuation and divisional application(s))), WO2010/079430 (and its national counterparts, more particularly its UScounterpart(s) (including the US continuation and divisionalapplication(s))).

TAL effectors have been discovered in phytopathogenic bacteria of thegenus Xanthomonas, and are key virulence factors of these bacteria. Onceinside the plant cell, they enter the nucleus, bind effector-specificDNA sequences and reprogram the host cell by mimicking eukaryotictranscription factors (Boch et al. 2009; Moscou et al. 2009). Anaturally-occurring TAL effector typically comprises:

-   -   a tandem repeat (or central domain), which is the direct tandem        repeat of adjacent amino acid units, wherein each unit of the        (tandem) repeat consists of 33, 34 or 35 amino acids, the N- to        C-ordered series of which determine the (specific) recognition        of a nucleotide sequence,    -   said tandem repeat being followed (in C-term) by a truncated        amino acid unit (usually, the truncation is at 20 amino acids),        which is not involved in the (specific) recognition of said        nucleotide sequence,    -   at least one Nuclear Localization Signal (NLS), and    -   an acidic transcriptional Activation Domain (AD) (cf. Boch et        al. 2009).

The number of (full-length) units of the tandem repeat of anaturally-occurring TAL effector (i.e., the number of amino acid units,which determine the (specific) recognition of the nucleotide sequence)may e.g., range from 8 to 39, more particularly from 10 to 33, usuallyfrom 12 to 27.

TAL effectors are highly conserved among the different bacterialspecies. Examples of TAL effectors, which derive from anaturally-occurring source, include AvrBs3 (from Xanthomonas campestrispv. vesicatoria), PthXo1 (from Xanthomonas oryzae pv. oryzae), AvrXa27(from Xanthomonas oryzae pv. oryzae), PthXo6 (from Xanthomonas oryzaepv. oryzae), PthXo7 (from Xanthomonas oryzae pv. oryzae).

The amino acid sequence of each (tandem) repeat unit is largelyinvariant within a TAL effector, with the exception of two adjacentamino acids, which are known as the Repeat Variable Diresidue (RVD), andwhich typically are at positions 12 and 13 within the repeat unit.

When a TAL effector repeat unit consists of 34 or 35 amino acids, theRVDs are at positions 12 and 13.

When a TAL effector repeat unit consists of 33 amino acids, the aminoacid that is at the second position in the RVD is missing (i.e., thevariable amino acid, which would have been at position 13, is missing).Hence, in such a situation, the RVD does not consist of two adjacentamino acids, but of only one amino acid. In accordance with theacknowledged terminology in the field of TAL effectors and TALENs, saidamino acid at position 12 is being referred to as a RVD, although it isnot followed by a variable amino acid at position 13.

Repeat units with different RVDs recognize different nucleotides, andthere is a direct correspondence between the RVDs in the repeat domainand the nucleotides in the target DNA sequence. Examples of RVDs and oftheir corresponding target nucleotides are given in Table 5 below.

TABLE 5 RVD nucleotide HD C NG T NI A NN G or A NS A or C or G N* C or THG T H* T IG T HA C ND C NK G HI C HN G NA G SN G or A YG T *denotes agap in the repeat sequence corresponding to a lack of the amino acidresidue at the second position of the RVD (e.g., when the repeatconsists of 33 amino acid, instead of 34 or 35 amino acids).

In accordance with the acknowledged terminology in the field of TALeffector and TALEN, each of the amino acid units that forms the tandemrepeat of a TAL effector or TALEN (i.e., each of the amino acid units,which determine the (specific) recognition of the nucleotide sequence)is being referred to as a (tandem) repeat unit, although the repeatunits of the same tandem repeat do not have the same sequence.

Engineered (or man-made or artificial) TAL effectors have been producedby modification of naturally-occurring TAL effectors.

For example, engineered (or man-made or artificial) TAL effectors havebeen produced by truncation of a naturally-occurring TAL effector, toproduce fragments of naturally-occurring TAL effector, which haveretained the DNA-binding function of the full length TAL effector. Moreparticularly, engineered (or man-made or artificial) TAL effectors havebeen produced by truncation of the acidic transcriptional ActivationDomain, to produce a fragment of a naturally-occurring TAL effector,which is devoid of the acidic transcriptional Activation Domain, butwhich has retained the DNA-binding function of the full length TALeffector.

Engineered (or man-made or artificial) TAL effectors have been producedby modification of the RVD sequence and/or by modification of the numberof repeat units of naturally-occurring TAL effectors or of fragmentsthereof, to recode them for defined target DNA sequences (cf. e.g., WO2011/072246 (and its national counterparts, more particularly its UScounterpart(s) (including the US continuation and divisionalapplication(s)), WO 2010/079430 (and its national counterparts, moreparticularly its US counterpart(s) (including the US continuation anddivisional application(s))).

TAL effectors have been used in genome editing (Bedell et al. 2012, Cadeet al. 2012, Chen et al. 2013, Qiu et al. 2013).

However, it is believed that TAL effectors and TALENs have not beenpreviously used for partial or full deletion of an (expanded) DNA tandemrepeat, more particularly for partial or full deletion of an (expanded)DNA tandem repeat, which is contained in a double-stranded DNA molecule,more particularly for partial or full deletion of an (expanded) DNAtandem repeat, which is contained in a double-stranded DNA molecule and,which forms a non-linear secondary structure, such as a hairpin, atriple helix or a tetraplex secondary structure.

It is also believed that TAL effectors and TALENs have not beenpreviously used for partial or full deletion of an (expanded) DNA tandemrepeat that is contained in a double-stranded DNA molecule, such asthose observed in pathological conditions.

The structure of the means of the application is especially adapted forpartial or full deletion of at least one DNA tandem repeat, moreparticularly for partial or full deletion of at least one DNA tandemrepeat in a double-stranded DNA, more particularly for partial or fulldeletion of at least one DNA tandem repeat, which is contained in adouble-stranded DNA and, which forms a non-linear secondary structure,such as a hairpin, a triple helix or a tetraplex secondary structure.

The structure of the means of the application is especially adapted forpartial or full deletion of at least one (expanded) DNA tandem repeatthat is contained in a double-stranded DNA molecule, such as thoseobserved in pathological conditions.

One of the means of the application is a DNA-binding polypeptide, whichbinds, or specifically binds, to a DNA nucleic acid comprising at leastone DNA tandem repeat, wherein said DNA-binding polypeptide comprises aTAL effector tandem repeat. A TAL effector tandem repeat consists ofadjacent amino acid units (of TAL effector tandem repeat), eachcontaining a Repeat Variable Diresidue (RVD) that determines recognitionof a nucleotide (cf. above).

According to an embodiment of the application, said TAL effector tandemrepeat units are immediately or directly adjacent to each other, i.e.,are contiguous.

Said DNA-binding polypeptide may further comprise at least one NuclearLocalization Signal (NLS), more particularly at least one NLS of a TALeffector.

The term “polypeptide” is herein intended in accordance with itsordinary meaning in the field of biology. The term “polypeptide”generally refers to a chain of amino acids linked by peptidic linkage.It does not imply any restriction in maximal length of the amino acidchain. As described below, a DNA-binding polypeptide of the applicationcomprises several units of TAL effector, and therefore has a minimallength that typically is above 50 amino acids, more particularly above60 amino acids, more particularly above 70 amino acids, moreparticularly above 100 amino acids, more particularly above 150 aminoacids, more particularly of at least 200 amino acids. The maximal lengthof a DNA-binding polypeptide of the application typically is below 2,000amino acids, more particularly below 1,500 amino acids, moreparticularly below 1,400 amino acids, more particularly below 1,000amino acids.

The DNA nucleic acid, to which the polypeptide of the application bindsor specifically binds, is a DNA nucleic acid that comprises at least oneDNA tandem repeat.

Said DNA nucleic acid can e.g., be a double-stranded DNA nucleic acid ora strand of a double-stranded DNA nucleic acid, more particularly adouble-stranded DNA nucleic acid, more particularly a chromosomaldouble-stranded DNA nucleic acid, more particularly a double-strandedDNA nucleic acid that is contained in a chromosome. Said double-strandedDNA nucleic acid can e.g., be a gene, more particularly a eukaryoticgene, more particularly a non-mammalian eukaryotic gene (e.g., a yeastgene) or a non-human mammalian gene (e.g., a rodent gene, a rat gene, amouse gene, a pig gene, a rabbit gene) or a human gene. According to anembodiment of the application, said at least one DNA nucleic acid is agene (or a strand of a gene), more particularly a human gene (or astrand of a human gene). Advantageously, said gene (more particularly,said human gene) is contained in a chromosome.

Said at least one DNA tandem repeat can be contained at any location(s)of said gene, e.g., in a promoter and/or in the 5′UTR and/or in at leastone exon and/or in at least one intron and/or in the 3′UTR of said gene.

In a DNA-binding polypeptide of the application, the ordered series ofRVDs formed by the RVDs respectively contained in said adjacent units ofTAL effector tandem repeat, in N- to C-orientation, is an ordered seriesof amino acids, which, according to the acknowledged RVD/nucleotidecorrespondence, determines the recognition of the 5′-3′ nucleotidesequence of a DNA target site contained in said DNA nucleic acid.

An acknowledged RVD/nucleotide correspondence is shown in Table 5 above.

According to an advantageous aspect of the application, a DNA-bindingpolypeptide of the application does not comprise the acidictranscriptional Activation Domain (AD) of a TAL effector. Such aDNA-binding polypeptide of the application does not have the function oftranscriptional activation that a naturally-occurring TAL effector has,but has retained the DNA-binding function of a full length TAL effector.

The sequence of said DNA target site consists of:

i. a fragment of the sequence of said at least one DNA tandem repeat, orii. a fragment of the sequence of said DNA nucleic acid, which startsoutside the sequence of said at least one DNA tandem repeat and whichends within the sequence of said at least one DNA tandem repeat, orconversely, which starts within the sequence of said at least one DNAtandem repeat and which ends outside the sequence of said at least oneDNA tandem repeat.

For the sake of concision, a DNA target site, the sequence of whichsatisfies feature i. above, will herein after be referred to as“non-overlapping DNA target site”, and a DNA target site, the sequenceof which satisfies feature ii. above, will herein after be referred toas “overlapping DNA target site”.

An example of non-overlapping DNA target site is the DNA target site ofSEQ ID NO: 10 or 11 (cf. example 1 and FIG. 1B).

According to an embodiment of the application, the DNA target site of aDNA-binding polypeptide of the application is a non-overlapping DNAtarget site.

An example of overlapping DNA target site is the DNA target site of SEQID NO: 4 or 5 (cf. example 1 and FIG. 1B).

For example, said DNA target site is:

-   -   either fully comprised within the DNA tandem repeat sequence:        cf. e.g., the DNA target site ^(5′)G(CTG)₄CT^(3′) (SEQ ID        NO: 10) shown underlined in FIG. 1B (right TALE binding domain),    -   or, is an overlapping site consisting of a fragment of the 5′ or        3′ end of the DNA tandem repeat sequence (wherein said fragment        contains the first nucleotide at said 5′ end or the last        nucleotide at said 3′ end respectively) and of a fragment of the        DNA sequence that is immediately adjacent to said 5′ or 3′ end        of DNA tandem repeat sequence outside said DNA tandem repeat        sequence respectively, i.e., a target site, which, for a portion        of it, is the 5′ or 3′ end (or extremity) of the DNA tandem        repeat sequence and for the rest of it the DNA sequence that is        immediately or directly adjacent to said 5′ or 3′ end of DNA        tandem repeat sequence (outside the DNA tandem repeat sequence):        cf. e.g., the DNA target site ^(5′)GTGATCCCCCCAGCA^(3′) (SEQ ID        NO: 4) shown underlined in FIG. 1B (non-split left TALE binding        domain), wherein the last five nucleotides, i.e.,        ^(5′)CAGCA^(3′) (SEQ ID NO: 6) is the sequence of the 5′ end of        the DNA tandem repeat and wherein ^(5′)GTGATCCCCC^(3′) (SEQ ID        NO: 7) is the DNA sequence that is immediately adjacent to the        5′ end of the DNA tandem repeat sequence outside said DNA tandem        repeat sequence (i.e., the gene sequence that is immediately or        directly adjacent to the 5′ end of ^(5′)CAGCA^(3′)).

According to an embodiment of the application, the DNA target site of aDNA-binding polypeptide of the application is an overlapping DNA targetsite.

A DNA tandem repeat occurs in a DNA nucleic acid, when a DNA sequenceunit (or pattern) of 2, 3, 4, 5 or more nucleotides is repeated, i.e.,the same DNA sequence unit of 2, 3, 4, 5 or more nucleotides isidentically repeated. Said DNA sequence unit can be any sequence of atleast two nucleotides, more particularly of at least two differentnucleotides.

When they relate to a DNA nucleic acid, the phrases “repeat”, “tandemrepeat”, “sequence unit(s)” and “unit(s)” (or equivalent or similarphrases) are given their respective general meaning of the field of DNAnucleic acids and DNA tandem repeats. For example, the nucleic acid

[SEQ ID NO: 23] GTGATCCCCCCAGCAGCAGCAGCAGCAGCAGCAG

contains a DNA tandem repeat consisting of eight copies of the sequenceunit CAG (said eight copies are shown underlined in SEQ ID NO: 23above).

According to an aspect of the application, said DNA sequence unitconsists of 2, 3, 4 or 5 nucleotides, wherein at least two nucleotidesof said unit are different nucleotides.

According to an aspect of the application, said DNA sequence unitconsists of 3, 4 or 5 nucleotides, wherein at least two nucleotides ofsaid unit or (at least) three nucleotides of said unit are differentnucleotides.

According to an aspect of the application, said DNA sequence unit isselected from the group consisting of ^(5′)CTG^(3′), ^(5′)CAG^(3′),^(5′)CAA^(3′), ^(5′)TTG^(3′), ^(5′)GAC^(3′), ^(5′)GTC^(3′),^(5′)CCTG^(3′), ^(5′)CAGG^(3′), ^(5′)ATTCT^(3′), ^(5′)AGAAT^(3′),^(5′)GCG^(3′), ^(5′)CGC^(3′), ^(5′)CGG^(3′) and ^(5′)CCG^(3′).

According to an aspect of the application, said DNA sequence unitconsists of 3 or 4 nucleotides, wherein at least two nucleotides of saidunit or (at least) three nucleotides of said unit are differentnucleotides.

According to an aspect of the application, said DNA sequence unit isselected from the group consisting of ^(5′)CTG^(3′), ^(5′)CAG^(3′),^(5′)CAA^(3′), ^(5′)TTG^(3′), ^(5′)GAC^(3′), ^(5′)GTC^(3′),^(5′)CCTG^(3′), ^(5′)CAGG^(3′), ^(5′)GCG^(3′), ^(5′)CGC^(3′),^(5′)CGG^(3′) and ^(5′)CCG^(3′).

According to an aspect of the application, said DNA sequence unitconsists of 3 nucleotides, wherein at least two nucleotides of said unitor the three nucleotides of said unit are different nucleotides.

According to an aspect of the application, said DNA sequence unit isselected from the group consisting of ^(5′)CTG^(3′), ^(5′)CAG^(3′),^(5′)CAA^(3′), ^(5′)TTG^(3′), ^(5′)GAC^(3′), ^(5′)GTC^(3′),^(5′)GCG^(3′), ^(5′)CGC^(3′), ^(5′)CGG^(3′)and ^(5′)CCG³.

The number of DNA sequence units that are repeated in said at least oneDNA tandem repeat is of at least 2 units. According to an aspect of theapplication, said number is of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51 or 52 units.

Within a DNA tandem repeat, the copies of the sequence unit are adjacentto each other. They can either be spaced apart from each other by only afew nucleotides, e.g., by less than 11, 10, 9, 8, 7, 6, 5, 4, 3, 2nucleotides, or can be directly adjacent to each other. According to anaspect of the application, said copies of DNA sequence unit are spacedapart from each other by only a few nucleotides, e.g., by less than 6,5, 4, 3, 2 nucleotides, or are directly adjacent to each other.According to an aspect of the application, said copies of DNA sequenceunit are directly adjacent to each other. For example, in theabove-mentioned nucleic acid of SEQ ID NO: 23, said copies of DNAsequence unit (i.e., the copies of the sequence unit CAG) are directlyadjacent to each other.

According to an aspect of the application, one DNA sequence unit (orpattern) does not consist of the same nucleotide. For example, thesequence unit CAG consists of three different nucleotides (C, A and G).

In the application, a DNA tandem repeat is a direct tandem repeat, i.e.,it is not an inverted tandem repeat: the order in which the nucleotidesare contained in one DNA sequence unit is conserved throughout the DNAtandem repeat.

When they relate to TAL effector or TALEN, the phrases “repeat”, “tandemrepeat”, “unit(s) of tandem repeat”, “TAL effector repeat unit(s)” or“repeat unit(s)” (or equivalent or similar phrases) are given theirrespective general meaning of the field of TAL effectors and TALENs. TALeffector repeat units are the amino acid units that form the tandemrepeat of the TAL effector, i.e., the amino acid units, which determinethe (specific) recognition of the nucleotide sequence of the DNA targetsite through the N- to C-ordered series of RVDs they respectivelycontain.

As mentioned above, the units, which are considered (and computed) asTAL effector repeat units, are those, which determine the recognition ofthe DNA target site by direct correspondence of the N- to C-orderedseries of RVDs they form with the 5′-3′ nucleotide sequence of the DNAtarget site (e.g., in accordance with Table 5 above). TAL effectorrepeat units do not include any TAL effector amino acid unit, whichwould not be involved in said (specific) recognition, such as e.g., theunit, which is in C-term of the central domain of a naturally-occurringTAL effector and, which is truncated at 20 amino acids.

The tandem repeat (or central domain) of a TAL effector consists ofadjacent amino acid sequence(s), which are known as the repeat units ofsaid TAL effector, and which each consist of a frame sequence in which aRVD is contained. Please see above for the description of the typicalstructure of a TAL effector, more particularly of a TAL effector repeatunit.

Said repeat units can be directly or non-directly adjacent to eachother; they are more particularly directly adjacent to each other.

For example, the polypeptide

[SEQ ID NO: 24] LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHGGKQALETVQRLLPVLCQAHGis a TAL effector tandem repeat consisting of eight (directly adjacent)copies of the repeat unit LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG [SEQ ID NO:25], wherein XX is the RVD and wherein the RVD is:

HD in the first repeat unit,

NG in the second repeat unit,

NI in the third repeat unit,

NN in the fourth repeat unit,

NS in the fifth repeat unit,

N* in the sixth repeat unit,

HG in the seventh repeat unit, and

H* in the eighth repeat unit,

(the first repeat unit is shown underlined in SEQ ID NO: 24, the RVDsare shown in bold characters in each of the eight units). In thisexample, the frame sequence of the TAL effector tandem repeat unit isthe sequence of SEQ ID NO: 25 (in this example, the frame sequence isthe same for each of the eight repeat units).

In a DNA-binding polypeptide of the application, the number of aminoacids that are contained in one TAL effector tandem repeat unit can be33, 34 or 35 (i.e., the same as in a naturally-occurring TAL effector),or can be lower, e.g., 29, 30, 31 or 32.

Hence, in a DNA-binding polypeptide of the application, the number ofamino acids that are contained in one TAL effector tandem repeat unitcan be an integer selected from 29-35, or from 30-35, or from 31-35, orfrom 32-35, or from 29-34, or from 30-34, or from 31-34, or from 32-34,or from 30-33, or from 30-34, or from 31-33, or from 32-33.

According to an aspect of the application, the number of amino acidsthat are contained in one repeat unit is 33, 34 or 35 (i.e., the same asin a naturally-occurring TAL effector), more particularly 34.

The TAL effector repeat units of a DNA-binding polypeptide of theapplication can each consist of the same number of amino acids, or canconsist of different numbers of amino acids. According to an embodimentof the application, the TAL effectors repeat units of a DNA-bindingpolypeptide of the application each consist of the same number of aminoacids, e.g., 33, 34 or 35 amino acids, e.g., 34 amino acids.

The N- to C-ordered series of TAL effector repeat units of a DNA-bindingpolypeptide of the application can be followed in C-term by a truncatedunit, which consists of less than 29 amino acids, more particularly aunit, which is truncated after the RVD (i.e., after the amino acid atposition 13), e.g., which is truncated immediately after the amino acidat position 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14or 13. An example of such a truncated unit is the unit of SEQ ID NO: 56(LTPQQVVAIASNGG), which can be viewed as a truncation of the TALeffector repeat unit of SEQ ID NO: 46(LTPQQVVAIASXXGGKQALETVQRLLPVLCQAHG) at amino acid position 14 withXX=NG. Such a truncated unit is not involved in the (specific)recognition of the nucleotide sequence of the DNA target site andtherefore is not considered as, and not computed as a TAL effectorrepeat unit.

As mentioned above, the units, which are considered (and computed) asTAL effector repeat units, are those, which determine the recognition ofthe DNA target site by direct correspondence of the N- to C-orderedseries of RVDs they form with the 5′-3′ nucleotide sequence of the DNAtarget site (e.g., in accordance with Table 5 above).

Units, which would not determine said recognition, such as theabove-mentioned truncated unit, are not considered, and are notcomputed, as a TAL effector repeat unit.

Hence, in the application, units, which consist of 29-35 amino acids asdescribed above, can be considered (and computed) as TAL effector repeatunits, whereas the above-mentioned truncated unit is not considered (andis not computed) as a TAL effector repeat unit.

The frame sequence of a TAL effector repeat unit is largely invariantamong the TAL effectors. Examples of (the frame sequence of a) TALeffector repeat unit comprise:

-   -   the sequence of SEQ ID NO: 25        (LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG; cf. example 1 below),    -   the sequence of SEQ ID NO: 46        (LTPQQVVAIASXXGGKQALETVQRLLPVLCQAHG; cf. example 1 below),    -   the sequence of SEQ ID NO: 55        (LTPEQVVAIASXXGGKQALETVQALLPVLCQAHG; cf. example 1 below),    -   the sequence of SEQ ID NO: 26        (LTPDQVVAIASXXGGKQALETVQRLLPVLCQDHG) wherein XX stands for the        RVD. Please note that, in a RVD sequence “XX”, the first X is an        amino acid, whereas the second X is an amino acid or is absent        (cf. e.g., N* or H* in Table 5 above).

Other examples of (the frame sequence of a) TAL effector repeat unitcomprise amino acid units, the respective sequences of which are variantsequences of at least one of the sequences of SEQ ID NOs: 25, 46, 55,26. Said variant sequences:

-   -   consist of 33, 34 or 35 amino acids,    -   are at least 50%, more particularly at least 51%, or at least        52%, or at least 53%, or at least 54%, or at least 55%, or least        55.5% identical to at least one of SEQ ID NOs: 25, 46, 55 and 26        over the whole length of said at least one SEQ ID sequence,    -   have retained the “XX” RVD sequence at positions 12 and 13, and    -   have retained the nucleotide recognition capacity of a TAL        effector tandem repeat unit (i.e., which determine the        recognition of a nucleotide through said “XX” RVD).

According to an aspect of the application, said XX is selected from thegroup consisting of HD, NG, NI, NN, NS, N*, HG, H*, IG, HA, ND, NK, HI,HN, NA, SN and YG. The symbol * denotes that the second X is missing(cf. e.g., Table 5 above).

A TAL effector tandem repeat can be:

-   -   formed by the same unit frame sequence, which is identically        repeated (like a homopolymer wherein only the RVDs vary), such        as illustrated above by the sequence of SEQ ID NO: 24, or can be    -   formed by different unit frame sequences, i.e., the TAL effector        repeat units do not all have the same frame sequence (like a        heteropolymer, e.g., as in the TAL effector tandem repeat        sequence coded by SEQ ID NO: 45 or SEQ ID NO: 54).

The TAL effector repeat units of a DNA-binding polypeptide of theapplication can have each different frame sequences. Nevertheless, a TALeffector tandem repeat of a DNA-binding polypeptide of the applicationgenerally consists of repeat units wherein at least one frame sequenceis identically repeated.

Although the TAL effector repeat units of a DNA-binding polypeptide canhave different frame sequences, said units are considered to be “repeatunits” in accordance with the acknowledged terminology in the field ofTALE effector and TALENs. Indeed, the sequence variation between (theframe sequences of) two different TAL effector units is low (cf. e.g.,the sequence variation between the 34aa-long sequences of SEQ IDs: 25,26, 46 and 55) and the function is conserved.

Hence, in a DNA-binding polypeptide of the application, the adjacentunits of TAL effector tandem repeat may for example comprise or consistof one or several copy(ies) of at least one sequence selected from thegroup consisting of SEQ ID NOs: 25, 26, 46, 55 and said variantsequences thereof, and/or comprise one or several copy(ies) of at leastone of the sequences of TAL effector tandem repeat units of theDNA-binding polypeptide, which is coded by the plasmid deposited at theCollection Nationale de Culture de Microorganismes (C.N.C.M.), Paris,France, under deposit number I-4804 or under deposit number I-4805.

The total number of the adjacent units forming the TAL effector tandemrepeat of a DNA-binding polypeptide of the application can be from 8 to39, usually from 10 to 33, 13 to 33, 13 to 34, 13 to 35, 14 to 33, 14 to34 or 14 to 35, for example from 12 to 27, 13 to 28, from 14 to 28, from14 to 22, from 15 to 21, e.g., 15, 16, 17, 18, 19, 20 or 21.

For example, in FIG. 1B, each of the two engineered TAL effectors bindsto a target DNA site consisting of 15 nucleotides (SEQ ID NO: 4 and 10,respectively); therefore, the number of repeat units of each of said twoengineered TAL effectors is 15.

Every combination of amino acid length of a TAL effector tandem repeatunit and of number of TAL effector tandem repeat units is hereinexplicitly encompassed, e.g., a number of amino acids of 29-35 per TALeffector tandem repeat unit and a number of 13-33 TAL effector tandemrepeat units per polypeptide, or a number of amino acids of 33-35 perTAL effector tandem repeat unit and a number of 12-27 TAL effectortandem repeat units per polypeptide.

A DNA-binding polypeptide of the application can be anon-naturally-occurring polypeptide, e.g., a man-made or artificial orengineered polypeptide.

According to an aspect of the application, a DNA-binding polypeptide ofthe application does not comprise the acidic transcriptional activationdomain (AD) of a TAL effector. According to this aspect of theapplication, a DNA-binding polypeptide of the application can be viewedas a fragment of TAL effector or of engineered TAL effector, which stillcomprises the tandem repeat and the NLS of said (engineered) TALeffector, and which is advantageously devoid of the acidictranscriptional activation domain (AD) of said TAL effector. Examples ofsuch fragments notably include the BamHI fragment of said TAL effector.

The total length of the TAL effector tandem repeat of a polypeptide ofthe application (i.e., the total length formed by the adjacent aminoacid units forming said TAL effector tandem repeat) can e.g., be above50, 60, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650or 700 amino acids and/or below 2,000, 1,800, 1,600, 1,500, 1,400,1,200, 1,000, 900, 800 or 750 amino acids.

Every combination of one of these minimal lengths and of one of thesemaximal lengths is herein explicitly encompassed, e.g., a length above50 and below 1,400 amino acids, or a length above 60 and below 1,400amino acids, or above 70 and below 1,400 amino acids, or above 1000 andbelow 1,400 amino acids, or above 150 and below 1,400 amino acids, orabove 200 and below 1,400 amino acids, or above 300 and below 1,500amino acids, or above 400 and below 1,200 amino acids, or above 500 andbelow 1,200 amino acids, or above 600 and below 1,200 amino acids, orabove 600 and below 1,000 amino acids, or above 650 and below 800 aminoacids, or above 700 and below 750 amino acids.

Hence, a DNA-binding polypeptide of the application can e.g., comprise aTAL effector tandem repeat, wherein the total number of adjacent aminoacid units forming said TAL effector tandem repeat is 8 to 39 (moreparticularly, 10 to 33, 13 to 33, 13 to 34, 13 to 35, 14 to 33, 14 to 34or 14 to 35, for example from 12 to 27, 13 to 28, from 14 to 28, from 14to 22, from 15 to 21, e.g., 15, 16, 17, 18, 19, 20 or 21), wherein eachof said adjacent units of said TAL effector tandem repeat is selectedfrom the group consisting of the sequences of SEQ ID NOs: 25, 26, 46, 55and said variant sequences thereof, and wherein the N- to C-orderedseries of RVDs formed by said adjacent repeat units determine therecognition of an overlapping DNA target site or of a non-overlappingDNA target site.

Said DNA-binding polypeptide may further comprise at least one NLS, moreparticularly at least one NLS of TAL effector.

According to an aspect of the application, the DNA target site, which isrecognized by the ordered series of RVDs of the TAL effector tandemrepeat units of a DNA-binding polypeptide of the application, consistsof 8 to 39 nucleotides, more particularly of 13 to 33, 13 to 34, 13 to35, 14 to 33, 14 to 34 or 14 to 35 nucleotides, for example of 13 to 28nucleotides, of 14 to 28 nucleotides, of 14 to 22 nucleotides, of 15 to21 nucleotides, e.g., of 15, 16, 17, 18, 19, 20 or 21 nucleotides.

According to an aspect of the application, said DNA target site consistsof a number of nucleotides, which is identical to the number of TALeffector tandem repeat units of said DNA-binding polypeptide.

According to an aspect of the application, a non-overlapping DNA targetsite as defined above is a fragment of said at least one DNA tandemrepeat, and comprises more than one copy of the DNA sequence unit ofsaid at least one DNA tandem repeat, more particularly at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 (adjacent or directly adjacent) copiesof the DNA sequence unit of said at least one DNA tandem repeat, moreparticularly at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 directlyadjacent copies of the DNA sequence unit of said at least one DNA tandemrepeat.

According to an aspect of the application, the number of copy(ies) ofDNA sequence unit in said fragment of said at least one DNA tandemrepeat is an integer.

According to an alternative or complementary aspect of the application,said copy number is not an integer, i.e., it is a number with decimals(more particularly a number with two decimals). For example, if the DNAsequence unit consists of 3 nucleotides, and if the fragment of the DNAtandem repeat that is contained in the non-overlapping DNA target siteconsists of five nucleotides, i.e., if it consists of one unit copy (3nucleotides) and (directly adjacent thereto) two thirds of another unitcopy (2 nucleotides), the copy number is 3/3+2/3=1.67, i.e., the copynumber is not an integer.

When it relates to a non-overlapping DNA target site, the expression“more than one copy” encompasses a copy number, which is or not aninteger, more particularly a copy number, which is more than one andless than two, such as a copy number of 1.67, as well as a copy numberof two and above.

A non-overlapping DNA target site can e.g., be a fragment of said atleast one DNA tandem repeat, which comprises or consists of more thanone copy of the DNA sequence unit, e.g., which comprises or consists of:

-   -   one copy, or several directly adjacent copies, of the DNA        sequence unit, and, directly adjacent thereto (in 5′ and/or in        3′),    -   zero, one fragment of said DNA sequence unit (in 5′ and/or in        3′), or two fragment(s) of said DNA sequence unit (one fragment        in 5′ and one fragment in 3′).

For example, if the DNA sequence unit of the DNA tandem repeat is^(5′)CTG^(3′), the sequence of the non-overlapping DNA target site cane.g., be ^(5′)G(CTG)₄CT^(3′) (SEQ ID NO: 10), i.e., a fragment of theDNA tandem repeat, which consists of four ^(5′)CTG^(3′) units ((CTG)₄)and, directly adjacent thereto, two fragments of DNA sequence unit(fragment G in 5′ and fragment CT in 3′).

An example of non-overlapping DNA target site is the DNA target site ofSEQ ID NO: 10 or of SEQ ID NO: 11 (cf. FIG. 1B). Hence, a DNA-bindingpolypeptide of the application can e.g., comprise a TAL effector tandemrepeat as defined above, wherein said units are selected from the groupconsisting of the sequences of SEQ ID NOs: 25, 26, 46, 55 and saidvariant sequences thereof, and wherein the N- to C-ordered series ofRVDs formed by the RVDs respectively contained in said units determinesthe recognition of a non-overlapping DNA target site as defined above,e.g., the DNA target site of SEQ ID NO: 10 or of SEQ ID NO: 11. Anexample of N- to C-ordered series of RVDs, which determines therecognition of the DNA target site of SEQ ID NO: 10 [^(5′)G(CTG)₄CT³],is: NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD and NG (cf.Table 5 above).

An example of TAL effector tandem repeat, which can be comprised in aDNA-polypeptide of the application and, which (specifically) binds to anon-overlapping DNA target site, is the polypeptide coded by thesequence of SEQ ID NO: 45 (cf. example 1 below), which (specifically)binds to the non-overlapping DNA target site of SEQ ID NO: 10.

An example of TAL effector tandem repeat, which can be comprised in aDNA-polypeptide of the application and, which (specifically) binds to anon-overlapping DNA target site, is the TAL effector tandem repeat codedby plasmid pCLS9996exp (C.N.C.M. deposit number I-4804), which(specifically) binds to the non-overlapping DNA target site of SEQ IDNO: 10.

The sequence of an overlapping DNA target site as defined above is thesequence of a fragment of said DNA nucleic acid, which, for a portion ofit, is within said at least one DNA tandem repeat [the “inside”portion], and which, for the remaining portion of it, is outside said atleast one DNA tandem repeat [the “outside” portion].

For example, if the sequence of the overlapping DNA target site is^(5′)GTGATCCCCCCAGCA^(3′) (SEQ ID NO: 4) within a DNA nucleic acidcomprising the (CAG)_(n) tandem repeat (cf. FIG. 1B), the portion of theDNA target site, which is within the DNA tandem repeat [the “inside”portion], consists of ^(5′)CAGCA^(3′) (SEQ ID NO: 6), and the remainingportion, which is outside said DNA tandem repeat [the “outside”portion], is ^(5′)GTGATCCCCC^(3′) (SEQ ID NO: 7), i.e., 10 nucleotides.

The portion of an overlapping DNA target site, which is within said atleast one DNA tandem repeat [the “inside” portion], is a fragment ofsaid at least one DNA tandem repeat, and consists of at least a fragmentof a copy of the DNA sequence unit of said at least one DNA tandemrepeat, more particularly of one, at least one or more than one copy ofthe DNA sequence unit of said at least one DNA tandem repeat, moreparticularly of two, at least two, three, at least three, four or atleast four (adjacent or directly adjacent) copies of the DNA sequenceunit of said at least one DNA tandem repeat.

According to an aspect of the application, said copy number is aninteger (e.g., in the DNA tandem repeat fragment ^(5′)CAGCAG^(3′) (SEQID NO: 35), the copy number is two (two ^(5′)CAG^(3′) units)).

According to an alternative or complementary aspect of the application,said copy number is not an integer i.e., it is a number with decimals(more particularly a number with two decimals). For example, if thesequence of the overlapping DNA target site is ^(5′)GTGATCCCCCCAGCA^(3′)(SEQ ID NO: 4) within a DNA nucleic acid comprising the (CAG)_(n) tandemrepeat (cf. FIG. 1B), the portion of the DNA target site, which iswithin the DNA tandem repeat [the “inside” portion], consists of^(5′)CAGCA^(3′) (SEQ ID NO: 6), i.e., consists of one DNA sequence unit(unit CAG) and (directly adjacent thereto) a fragment of the DNAsequence unit (CA), i.e., the “inside” portion consists of one unit copy(unit CAG) and, directly adjacent thereto, two thirds of another unitcopy (CA), the copy number is 1+2/3=1.67, i.e., the copy number is notan integer.

When it relates to the portion of an overlapping DNA target site, whichis within the DNA tandem repeat, the expression “more than one copy”encompasses a copy number, which is or not an integer, more particularlya copy number, which is more than one and less than two, such as a copynumber of 1.67, as well as a copy number of two and above.

More particularly, the portion of an overlapping DNA target site, whichis within said at least one DNA tandem repeat [the “inside” portion], isa fragment of said at least one DNA tandem repeat, which comprises orconsists of more than one copy of the DNA sequence unit, e.g., whichcomprises or consists of:

-   -   one copy, or several directly adjacent copies, of the DNA        sequence unit, and, directly adjacent thereto (in 5′ and/or in        3′),    -   zero, one fragment of said DNA sequence unit (in 5′ and/or in        3′), or two fragment(s) of said DNA sequence unit (one fragment        in 5′ and one fragment in 3′).

For example, if the DNA sequence unit of the DNA tandem repeat is^(5′)CTG^(3′), the sequence of the portion of the overlapping DNA targetsite, which is within said at least one DNA tandem repeat [the “inside”portion], can e.g., be ^(5′)G(CTG)₄CT^(3′) (SEQ ID NO: 10), i.e., afragment of the DNA tandem repeat, which consists of four ^(5′)CTG^(3′)units ((CTG)₄) and, directly adjacent thereto, two fragments of DNAsequence unit (fragment G in 5′ and fragment CT in 3′).

The portion of an overlapping DNA target site, which is outside said DNAtandem repeat [the “outside” portion], consists of at least 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 nucleotide(s), for example of at least 5 nucleotidesor of more than 5 nucleotides, more particularly of at least 6, 7, 8, 9or 10 nucleotides.

Any combination of:

-   -   said number of nucleotides of the “outside” (or first) portion        and of    -   said copy number of DNA repeat unit(s) of the “inside” (or        second) portion is herein explicitly encompassed, e.g., an        overlapping DNA target site, comprising at least one or more        than one copy of the DNA repeat unit of said at least one DNA        tandem repeat (cf. above) and comprising an “outside” portion of        at least 5, 6 or 7 nucleotides.

Alternatively or complementarily, the sequence of an overlapping DNAtarget site as defined above can be viewed as the sequence of a fragmentof said DNA nucleic acid, which comprises or consists of:

-   -   a. a sequence comprising, or consisting of, at least one, or        more than one, copy of the DNA sequence unit (cf. above), and,        directly adjacent thereto, in 5′ or in 3′,    -   b. a sequence, which is of at least 5 nucleotides or of more        than five nucleotides, and which differs from the sequence of a.

Alternatively or complementarily, the sequence of an overlapping DNAtarget site can be viewed as the sequence of a fragment of said DNAnucleic acid, which comprises, but does not consist of, a fragment ofsaid at least one DNA tandem repeat, wherein the copy number of said DNAsequence unit in said fragment of said at least one DNA tandem repeat isat least one or more than one, more particularly more than one.

More particularly, said fragment of said DNA nucleic acid furthercomprises another sequence, which is of at least five or of more thanfive nucleotides, and which is directly adjacent in 5′ or in 3′ to saidfragment of said at least one DNA tandem repeat. More particularly, theend of said sequence of at least five or of more than five nucleotides,which is directly linked to said fragment of said at least one DNAtandem repeat, is a sequence (e.g., of the same length as said DNAsequence unit, but) which differs from the sequence of said DNA sequenceunit.

Alternatively or complementarily, the sequence of an overlapping DNAtarget site can be viewed as the sequence of a fragment of said DNAnucleic acid, which consists of:

-   -   a. a nucleotide sequence, which is a fragment of said at least        one DNA tandem repeat, wherein the copy number of said DNA        sequence unit in said fragment of said at least one DNA tandem        repeat is more than one (said more than one copy being adjacent        or directly adjacent to each other, more particularly directly        adjacent to each other), and    -   b. a nucleotide sequence of at least five or of more than five        nucleotides, which differs from said sequence of a., and which        is directly linked, in 5′ or in 3′, to said nucleotide sequence        of a.,    -   wherein the end of said nucleotide sequence of b., which is        directly linked to said nucleotide sequence of a., is a sequence        (e.g., of the same length as said DNA sequence unit, but) which        differs from the sequence of said DNA sequence unit.

Alternatively or complementarily, an overlapping DNA target site can beviewed as a fragment of said DNA nucleic acid, which consists of aportion, which is outside the DNA tandem repeat [the “outside” portion]and of a portion, which is inside the DNA tandem repeat [the “inside”portion], wherein the nucleotide length of said “outside” portion ismore than 20%, more particularly more than 30%, more particularly morethan 40%, more particularly more than 45%, more particularly more than50%, more particularly more than 55%, more particularly more than 60%(but less than 100%) of the total length of said (full-length) DNAtarget site.

For example, if the sequence of the overlapping DNA target site is^(5′)GTGATCCCCCCAGCA3′ (SEQ ID NO: 4) within a DNA nucleic acidcomprising the (CAG)_(n) tandem repeat (cf. FIG. 1B), the portion of theDNA target site, which is outside the DNA tandem repeat, consists of^(5′)GTGATCCCCC^(3′) (SEQ ID NO: 7), i.e., consists of 10 nucleotides,whereas the DNA target site consists of 15 nucleotides; hence, theportion of the overlapping DNA target site, which is outside the DNAtandem repeat, consists of a number of nucleotides, which is(10/15×100=) 66.7%, i.e., of more than 60% (but less than 100%) of thetotal number of nucleotides of the DNA target site.

Advantageously, an overlapping DNA target site is a fragment of said DNAnucleic acid, which consists of a portion, which is outside the DNAtandem repeat [the “outside” portion] and of a portion, which is insidethe DNA tandem repeat [the “inside” portion], wherein the nucleotidelength of said “outside” portion is more than 40%, more particularlymore than 45%, more particularly more than 50%, more particularly morethan 55%, more particularly more than 60% (but less than 100%) of thetotal length of said (full-length) DNA target site.

Alternatively or complementarily, an overlapping DNA target site is afragment of said DNA nucleic acid, which consists of a portion, which isoutside the DNA tandem repeat [the “outside” portion] and of a portion,which is inside the DNA tandem repeat [the “inside” portion], whereinthe nucleotide length of said “inside” portion is less than 80%, moreparticularly less than 70%, more particularly less than 60%, moreparticularly less than 55%, more particularly less than 50%, moreparticularly less than 45%, more particularly less than 40% (but morethan 0% or more than 1%) of the total length of said (full-length) DNAtarget site. For example, if the sequence of the overlapping DNA targetsite is ^(5′)GTGATCCCCCCAGCA^(3′) (SEQ ID NO: 4) within a DNA nucleicacid comprising the (CAG)_(n) tandem repeat (cf. FIG. 1B), the portionof the DNA target site, which is inside the DNA tandem repeat, consistsof ^(5′)CAGCA^(3′) (SEQ ID NO: 6), i.e., of 5 nucleotides, whereas theDNA target site consists of 15 nucleotides; hence, the portion of theoverlapping DNA target site, which is inside the DNA tandem repeat,consists of a number of nucleotides, which is (5/15×100=) 33.3%, i.e.,of less than 40% (but more than 0% or more than 1%) of the total numberof nucleotides of the DNA target site.

Advantageously, an overlapping DNA target site is a fragment of said DNAnucleic acid, which consists of a portion, which is outside the DNAtandem repeat [the “outside” portion] and of a portion, which is insidethe DNA tandem repeat [the “inside” portion], wherein the nucleotidelength of said “inside” portion is less than 60%, more particularly lessthan 55%, more particularly less than 50%, more particularly less than45%, more particularly less than 40% (but more than 0% or more than 1%)of the total length of said (full-length) DNA target site.

Alternatively or complementarily, an overlapping DNA target site is afragment of said DNA nucleic acid, which comprises, but does not consistof, a fragment of said at least one DNA tandem repeat, wherein thenucleotide length of said at least one DNA tandem repeat is more than10% and less than 80%, more particularly more than 15% and less than70%, more particularly more than 20% and less than 60%, moreparticularly more than 20% and less than 50%, more particularly morethan 20% and less than 40%, of the total nucleotide length of said DNAtarget site.

An example of overlapping DNA target site is the DNA target site of SEQID NO: 4 or of SEQ ID NO: 5 (cf. FIG. 1B).

Hence, a DNA-binding polypeptide of the application can e.g., comprise aTAL effector tandem repeat as defined above, wherein said adjacent unitsare selected from the group consisting of the sequences of SEQ ID NOs:25, 26, 46, 55 and said variant sequences thereof, and wherein the N- toC-ordered series of RVDs formed by said adjacent units determine therecognition of the (overlapping) DNA target site of SEQ ID NO: 4 or ofSEQ ID NO: 5.

An example of N- to C-ordered series of RVDs, which determine therecognition of the DNA target site of SEQ ID NO: 4[^(5′)GTGATCCCCCCAGCA^(3′)], is NN; NG; NN; NI; NG; HD; HD; HD; HD; HD;HD; NI; NN; HD and NI (cf. Table 5 above).

An example of TAL effector tandem repeat, which can be comprised in aDNA-polypeptide of the application, is the polypeptide coded by thesequence of SEQ ID NO: 54 (cf. example 1 below), which (specifically)binds to the overlapping DNA target site of SEQ ID NO: 4.

An example of TAL effector tandem repeat, which can be comprised in aDNA-polypeptide of the application, is the TAL effector tandem repeatcoded by plasmid pCLS16715 (C.N.C.M. deposit number I-4805), which(specifically) binds to the overlapping DNA target site of SEQ ID NO: 4.

According to an aspect of the application, the sequence of the DNAtarget site that is recognized by the ordered series of RVDs of aDNA-binding polypeptide of the application is immediately preceded in 5′by the nucleotide T.

Indeed, it has been observed that the presence of the nucleotide Tdirectly adjacent to the 5′ end (or extremity) of the DNA target sitemight be advantageous to adequately or efficiently bind to anaturally-occurring DNA target.

For example, the DNA target site of SEQ ID NO: 10 (cf. right TALEbinding domain of FIG. 1B) is immediately preceded in 5′ by thenucleotide T. The DNA target site of SEQ ID NO: 4 (cf. the non-splitleft TALE binding domain of FIG. 1B) also is immediately preceded in 5′by the nucleotide T.

Said preceding T is not part of the DNA target site (the RVDs of theTALE effector do not determine the recognition of said T), but isbelieved to improve the stability of the binding.

The (at least one) DNA nucleic acid, to which the DNA-bindingpolypeptide of the application binds, or specifically binds, can e.g, bea double-stranded DNA nucleic acid or a strand of a double-stranded DNAnucleic acid.

Said DNA strand can be isolated from the other strand of thedouble-stranded DNA nucleic acid, whereby forming a single-strandedmolecule, or can still be contained in said double-stranded DNA nucleicacid molecule (i.e., be still in duplex with its complementary strand).Advantageously, said DNA strand is still contained in saiddouble-stranded DNA nucleic acid molecule.

Hence, the DNA nucleic acid, to which the DNA-binding polypeptide of theapplication binds, or specifically binds, advantageously is adouble-stranded DNA nucleic. When said DNA nucleic acid is adouble-stranded DNA nucleic acid, the DNA-binding polypeptide of theapplication binds to one of the two strands of the double-stranded DNAnucleic acid.

Said double-stranded DNA nucleic acid can e.g., be a chromosomal DNAnucleic acid, more particularly a chromosomal double-stranded DNAnucleic acid, more particularly a double-stranded DNA nucleic acid thatis contained in a chromosome.

Said double-stranded DNA nucleic acid can e.g., be a gene, moreparticularly a eukaryotic gene, more particularly a non-mammalianeukaryotic gene (e.g., a yeast gene) or a non-human mammalian gene(e.g., a rodent gene, a rat gene, a mouse gene, a pig gene, a rabbitgene) or a human gene. According to an embodiment of the application,said at least one DNA nucleic acid is a gene, more particularly a humangene. Advantageously, said gene (more particularly, said human gene) isa chromosomal gene, more particularly a gene that is contained in achromosome, more particularly a gene that is contained in a humanchromosome.

According to an aspect of the application, the DNA nucleic acid, towhich the DNA-binding polypeptide of the application binds, orspecifically binds, is a double-stranded DNA nucleic, wherein at leastone of its two strands contains nucleotide(s) T in the sequence of theDNA tandem repeat (i.e., in at least one of said two DNA strands, theunit of the DNA tandem repeat contains at least one nucleotide T).According to this aspect of the application, the DNA sequence unit thatis repeated in the sequence of said T-containing DNA tandem repeat cane.g., be selected from the group consisting of ^(5′)CTG^(3′),^(5′)TTG^(3′), ^(5′)GTC^(3′), ^(5′)CCTG^(3′), ^(5′)ATTCT^(3′) and^(5′)AGAAT^(3′).

According to an aspect of the application, the DNA nucleic acid, towhich the DNA-binding polypeptide of the application binds, orspecifically binds, is a double-stranded DNA nucleic, wherein only oneof its two strands contains the nucleotide T in the sequence of said atleast one DNA tandem repeat (i.e., in one of said two DNA strands, theunit of the DNA tandem repeat contains at least one nucleotide T,whereas in the other of said two DNA strands, the unit of the DNA tandemrepeat does not contain any nucleotide T). According to this aspect ofthe application, the DNA sequence unit that is repeated in the sequenceof said T-containing DNA tandem repeat can e.g., be selected from thegroup consisting of ^(5′)CTG^(3′), ^(5′)TTG^(3′), ^(5′)GTC^(3′)and^(5′)CCTG^(3′). The DNA-binding polypeptide of the application may bindto the strand that contains said nucleotide T, or may bind to the otherstrand. Please see FIG. 1B, which illustrates a human gene, wherein onlyone of its two strands contains the nucleotide T in the sequence of theDNA tandem repeat (i.e., the human gene coding for DM1, which comprisesthe (CAG)n tandem repeat in one strand and the (CTG)n tandem repeat inthe complementary strand): as described in example 1 below, a firstDNA-binding polypeptide of the application (i.e., the left-hand TALEN ofexample 1 below) binds to the strand containing the (CAG)n repeat at anoverlapping DNA binding site (e.g., SEQ ID NO: 4), whereas a secondDNA-binding polypeptide of the application, which is different from saidfirst DNA-binding polypeptide of the application, (i.e., the right-handTALEN of example 1 below) binds to the (complementary) strand containingthe (CAG)n repeat at a non-overlapping DNA binding site (e.g., SEQ IDNO: 10).

According to an aspect of the application, the DNA nucleic acid, towhich the DNA-binding polypeptide of the application binds, orspecifically binds, is a double-stranded DNA nucleic, wherein each ofits two strands contains the nucleotide T in the sequence of the DNAtandem repeat (i.e., in one of said two DNA strands, the unit of the DNAtandem repeat contains at least one nucleotide T, and in the other ofsaid two DNA strands, the unit of the DNA tandem repeat also contains atleast one nucleotide T). According to this aspect of the application,the DNA sequence unit that is repeated in the sequence of said(T-containing) DNA tandem repeat can e.g., be selected from the groupconsisting of ^(5′)ATTCT^(3′)and ^(5′)AGAAT^(3′). According to anadvantageous aspect of the application, the sequence of said at leastone DNA tandem repeat can form a non-linear secondary structure, such asa hairpin, a triple helix or a tetraplex secondary structure.

According to an advantageous aspect of the application, said DNA nucleicacid can be any DNA nucleic acid, more particularly any double-strandedDNA nucleic acid (more particularly any human double-stranded DNAnucleic acid), more particularly any gene (more particularly any humangene), which is involved in a neurological and/or muscular and/orskeletal disorder or disease and/or in a disorder or disease involvingat least one (abnormally-expanded) DNA tandem repeat, more particularlyin a neurological and/or muscular and/or skeletal disorder or diseaseinvolving at least one (abnormally-expanded) DNA tandem repeat.

Examples of such disorders and diseases, as well as of the genes thatare respectively involved in said disorders and diseases are given inTables 6, 7 and 8 below. Table 8 below shows examples of the averagenumber of DNA tandem repeat units that is observed in a healthy subject(normal average range of repeat units).

TABLE 6 DISEASE or DISORDER Acronym or Phenotype MIM abbreviation Namenumber (*) DM1 Myotonic dystrophy type 1 160900 SCA8 Spinocerebellarataxia 8 608768 SCA12 Spinocerebellar ataxia 12 604326 HDL2 Huntington'sdisease-like 2 606438 SBMA Spinal and bulbar muscular atrophy 313200 (orKennedy disease) HD Huntington's disease 143100 DRPLADentatorubral-pallidouysian atrophy 125370 SCA1 Spinocerebellar ataxia 1164400 SCA2 Spinocerebellar ataxia 2 183090 SCA3 Spinocerebellar ataxia3 109150 (Machado-Joseph disease) SCA6 Spinocerebellar ataxia 6 183086SCA7 Spinocerebellar ataxia 7 164500 SCA17 Spinocerebellar ataxia 17607136 PSACH Pseudoachondroplasia 177170 DM2 Myotonic dystrophy 2 602668SCA10 Spinocerebellar ataxia 10 603516 SPD1 Synpolydactyly 186000 OPMDOculopharyngeal muscular dystrophy 164300 CCD Cleidocranial dysplasia119600 HPE5 Holoprosencephaly 5 609637 HFG Hand-Foot-Genital syndrome140000 syndrome BPES Blepharophimosis, epicanthus inversus, 110100 andptosis EIEE1 Epileptic encephalopathy, early 308350 infantile, 1 FRAXAFragile X syndrome 300624 FXTAS X tremor/ataxia syndrome 300623 FRAXEMental retardation, X-linked, 309548 associated with fragile site FRAXE

TABLE 7 GENE DISEASE or Gene ID DISORDER MIM of the (acronym or Name ofthe protein number sequence abbreviation) encoded by the gene (*) (§)DM1 DMPK (dystrophia myotonia protein 605377 1762 kinase) SCA8 ATXN8;protein-coding strand 613289 724066 SCA8 ATXN8; non-protein codingstrand 603680 6315 (ATXN8OS) SCA12 PPP2R2B (regulatory subunit B of604325 5521 protein phosphatase 2) HDL2 JPH3 (junctophilin-3) 60526857338 SBMA AR (androgen receptor) 313700 367 HD HTT (huntingtin) 6130043064 DRPLA ATN1 (atrophin 1) 607462 1822 SCA1 ATXN1 (ataxin-1) 6015566310 SCA2 ATXN2 (ataxin-2) 601517 6311 SCA3 ATXN3 (ataxin-3) 607047 4287SCA6 CACNA1A (calcium channel, 601011 773 voltage-dependent, P/Q type,alpha 1A subunit) SCA7 ATXN7 (ataxin-7) 607640 6314 SCA17 TBP (TATAbox-binding protein) 600075 6908 PSACH COMP 600310 1311 DM2 ZNF9(zinc-finger protein) 116955 7555 SCA10 ATXN10 (ataxin-10) 611150 25814SPD1 HOXD13 (homeobox D13) 142989 3239 OPMD PABN1 (poly(A)-bindingprotein-2) 602279 8106 CCD RUNX2 (runt-related transcription 600211 860factor 2) HPE5 ZIC2 (zinc-finger protein of 603073 7546 cerebellum 2)HFG HOXA13 (homeobox A13) 142959 3209 syndrome BPES FOXL2 605597 668EIEE1 ARX (homeobox gene) 300382 170302 FRAXA FMR1 (fragile X mentalretardation 309550 2332 1) FXTAS FMR1 (fragile X mental retardation309550 2332 1) FRAXE AFF2 300806 309548

TABLE 8 REPEAT UNIT Coding = C Normal Comple- [encoded amino averageDISEASE or mentary acids] Non- range of DISORDER 5′-3′ strand 5′-3′Coding = NC repeat units DM1 (CTG)n (CAG)n NC 5-37 SCA8 (non- (CTG)n(CAG)n NC 15-50  protein coding strand, ATXN8OS) SCA8 (CAG)n (CTG)n C[polyGln] (ATXNS- coding strand) SCA12 (CAG)n (CTG)n NC 7-32 HDL2 (CAG)n(CTG)n NC 6-28 SBMA (CAG)n (CTG)n C [polyGln] 10-36  HD (CAG)n (CTG)n C[polyGln] 9-36 DRPLA (CAG)n (CTG)n C [polyGln] 7-25 SCA1 (CAG)n (CTG)n C[polyGln] 6-39 SCA2 (CAG)n (CTG)n C [polyGln] 13-31  SCA3 (CAG)n (CTG)nC [polyGln] 13-44  SCA6 (CAG)n (CTG)n C [polyGln] 4-18 SCA7 (CAG)n(CTG)n C [polyGln] 4-35 SCA17 (CAG)n (CTG)n C [polyGln] 25-44  SCA17(CAA)n (TTG)n C [polyGln] 25-44  PSACH (GAC)n (GTC)n C [polyAsp]  5 DM2(CCTG)n (CAGG)n NC ≦30  SCA10 (ATTCT)n (AGAAT)n NC 10-29  SPD1 (GCG)n(CGC)n C [polyAla] 15 OPMD (GCG)n (CGC)n C [polyAla]  6 CCD (GCG)n(CGC)n C [polyAla] 17 HPE5 (GCG)n (CGC)n C [polyAla] 15 HFG (GCG)n(CGC)n C [polyAla] 18 syndrome BPES (GCG)n (CGC)n C [polyAla] 14 EIEE1(GCG)n (CGC)n C [polyAla] 10-16  FRAXA (CGG)n (GCG)n NC 6-52 FXTAS(CGG)n (GCG)n NC 6-52 FRAXE (CCG)n (CGG)n NC 4-39(*) MIM number of the Online Mendelian Inheritance in Man® (OMIM®)database. OMIM® is authored and edited at the McKusick-Nathans Instituteof Genetic Medicine, Johns Hopkins University School of Medicine,U.S.A., under the direction of Dr. Ada Hamosh; please seehttp://www.omim.org/ as well as McKusick, V. A. 1998 (MendelianInheritance in Man; A Catalog of Human Genes and Genetic Disorders,Baltimore, Md., U.S.A., Johns Hopkins University Press, ISBN0-8018-5742-2).(§) Gene ID as available from NCBI (National Center for BiotechnologyInformation, U.S. National Library of Medicine, 8600 Rockville Pike,Bethesda Md., 20894, U.S.A.) [http://www.ncbi.nlm.nih.gov/].

According to an aspect of the application, the DNA nucleic acid, towhich the DNA-binding polypeptide of the application binds, orspecifically binds, is a gene, more particularly the human gene, codingfor DMPK, ATXN8, PPP2R2B, JPH3, AR, HTT, ATN1, ATXN1, ATXN2, ATXN3,CACNA1A, ATXN7, TBP, COMP, ZNF9, ATXN10, HOXD13, PABN1, RUNX2, ZIC2,HOXA13, FOXL2, ARX, FMR1 or AFF2 (wherein said gene comprises said atleast one DNA tandem repeat).

According to an aspect of the application, said DNA nucleic acid is agene, more particularly the human gene, coding for DMPK, ATXN8, PPP2R2B,JPH3, AR, HTT, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, COMP,ZNF9 or ATXN10 (wherein said gene comprises said at least one DNA tandemrepeat).

More particularly, said DNA nucleic acid can be a gene, moreparticularly the human gene, coding for DMPK (wherein said genecomprises said at least one DNA tandem repeat).

According to an aspect of the application, the number of DNA tandemrepeat units contained in said human DNA nucleic acid is above theaverage normal range, e.g., above the range that is observed in ahealthy subject, e.g., above the average normal range of repeat unitsrespectively indicated in Table 8 above (i.e., above 37 for the DM1disease or disorder, above 50 for the SCA8 disease or disorder, above 32for the SCA12 disease or disorder, etc.).

The DNA-binding polypeptide of the application can be bound to said DNAnucleic acid. More particularly, the DNA-binding polypeptide of theapplication can be bound to said DNA nucleic acid in vitro or in an invitro cell.

The DNA-binding polypeptide of the application can be directly orindirectly linked to at least one endonuclease monomer, or to at leastone fragment of endonuclease monomer, wherein said fragment ofendonuclease monomer still comprises the catalytic domain of saidendonuclease monomer.

More particularly, the DNA-binding polypeptide of the application can bedirectly or indirectly linked to one endonuclease monomer, or onefragment of endonuclease monomer, wherein said fragment of endonucleasemonomer still comprises the catalytic domain of said endonucleasemonomer.

The linkage of said (at least) one endonuclease monomer or fragmentthereof to said DNA-binding polypeptide is such that it does not impedethe endonuclease activity or function of said at least one endonucleasemonomer or at least fragment thereof.

The resulting structure can be viewed as and functions as a TALENmonomer.

In the application, the phrase “endonuclease” and the phrase “catalyticdomain” (or equivalent or similar phrases) are given their respectiveordinary meaning in the field of enzymology, more particularly in thefield of enzymology for biotechnological applications. An endonucleasecan e.g., be defined as an enzyme that cleaves phosphodiester bond(s)within polynucleotide chain(s). The catalytic domain of an endonucleasecan e.g., be defined as the region of said endonuclease, which containsthe catalytic function of the endonuclease.

In the application, the phrase “catalytic domain” (or an equivalent orsimilar phrase) can be understood as meaning “cleavage domain”, i.e.,the portion of the endonuclease, which causes the cleavage of thepolynucleotide chain(s).

In the application, the phrase “linked” (or an equivalent or similarphrase) encompasses direct linkage, as well as indirect linkage. Itencompasses any chemical linkage, more particularly covalent linkage,more particularly divalent covalent linkage.

Appropriate endonucleases notably comprise endonucleases, which functionas multimers, more particularly as dimers.

A dimeric endonuclease is an endonuclease, which is formed by twomonomers, the dimerization of which is required to cleave the target DNAdouble strand. Each monomer of a dimeric endonuclease comprises acatalytic domain.

Examples of such dimeric endonucleases notably include the FokIendonuclease (Christian et al. 2010, Li et al. 2011, WO 94/18313 and itsnational counterparts more particularly its US counterpart(s), includingthe US continuation and divisional application(s)), WO 95/09233 and itsnational counterparts more particularly its US counterpart(s), includingthe US continuation and divisional application(s)). An example of thesequence of a Fold endonuclease and of its catalytic domain is availableunder GENBANK accession number A32861. When the endonuclease is amultimeric endonuclease, more particularly a dimeric endonuclease, theDNA-binding polypeptide of the application is advantageously linked toonly one of said endonuclease monomers (advantageously in only oneexemplar).

Appropriate endonucleases also comprise monomeric endonucleases. Amonomeric endonuclease cleaves DNA, when it is used as single monomer aswell as when it is used in a pair of monomeric endonucleases. Examplesof monomeric endonucleases include I-TevI, which is the homingendonuclease member of the GIY-YIG protein family. Examples of fragmentsof I-TevI, which still comprise the endonuclease catalytic domain,include the I-TevI fragment, which consists of the N-terminal 183residues of wild-type I-TevI and a linker of 5 amino acids, e.g., thelinker QGPSG [SEQ ID NO: 22] (Beurdeley et al. 2013). When theendonuclease is a monomeric endonuclease, the DNA-binding polypeptide ofthe application is advantageously linked to said endonuclease monomer inonly one exemplar.

Examples of endonucleases also include non-naturally occurringendonuclease, e.g., a non-naturally occurring endonuclease, whichderives from a naturally-occurring endonuclease, more particularly froma naturally-occurring dimeric endonuclease, by amino acid mutation(s)(e.g., by amino acid replacement(s) and/or deletion(s) and/oraddition(s), more particularly by amino acid replacement(s)). Forexample, said non-naturally occurring restriction endonuclease can be a(homo- or hetero-) dimer, which differs from the FokI dimer by aminoacid mutation(s) in the catalytic domain of one or each of the two Foldmonomers. The number of amino acid mutation(s) per mutated Fold monomercan e.g., be of three to six. For example, said amino acid mutation(s)can be three to six mutations selected from positions 483, 486, 487,490, 499 and 538 of the catalytic domain as described in cf. WO2012/015938 and its national counterparts, including its US nationalcounterpart(s).

Advantageously, the DNA-binding polypeptide of the application is linkedto only one endonuclease monomer (advantageously at only one exemplar).

In the application, the phrase “an endonuclease monomer” (or anequivalent or a similar phrase) encompasses a monomer of a dimericendonuclease, as well as the monomer of a monomeric endonuclease.

For medical applications, more particularly for applications relating totreatment and/or palliation and/or prevention of diseases or disorders,a dimeric endonuclease might be preferred to a pair of monomericendonucleases, because a monomeric endonuclease might induce off-targetsingle-strand cleavage.

According to an embodiment of the application, the endonuclease is adimeric (naturally-occurring or non naturally-occurring) endonuclease,such as FokI.

A fragment from an endonuclease monomer, which still comprises thecatalytic domain of the endonuclease monomer, can also be used.

Said fragment can be a fragment of a monomer of a dimeric endonuclease,or a fragment of a monomeric endonuclease (said endonuclease beingnaturally-occurring or non-naturally-occurring).

An example of a FokI endonuclease monomer is the sequence of SEQ ID NO:49 (cf. example 1 below).

Examples of a DNA-binding polypeptide of the application, which isdirectly or indirectly linked to an endonuclease monomer or to afragment of endonuclease monomer, and which (specifically) binds to anon-overlapping DNA target site (i.e., the DNA target site of SEQ ID NO:10) include:

-   -   the polypeptide coded by the sequence of SEQ ID NO: 39, and    -   the polypeptide coded by plasmid pCLS9996exp (C.N.C.M. deposit        number I-4804).

Examples of a DNA-binding polypeptide of the application, which isdirectly or indirectly linked to an endonuclease monomer or to afragment of endonuclease monomer, and which (specifically) binds to anoverlapping DNA target site (i.e., the DNA target site of SEQ ID NO: 4),include:

-   -   the polypeptide coded by the sequence of SEQ ID NO: 50, and    -   the polypeptide coded by plasmid pCLS16715 (C.N.C.M. deposit        number I-4805).

A DNA-binding polypeptide of the application may further comprise adetection label or a selection marker, such as kanamycin or a knockoutleucine synthesis gene (e.g., LEU2) (cf. example 1 below).

The application also relates to a set comprising a first DNA-bindingpolypeptide and a second DNA-binding polypeptide, wherein only one, oreach one, of said first and second DNA-binding polypeptides is aDNA-binding polypeptide of the application. Said first DNA-bindingpolypeptide is different from said second DNA-binding polypeptide. Saidset can be herein referred to as the “polypeptide set of theapplication”.

A polypeptide set of the application is:

-   -   a set comprising a first DNA-binding polypeptide and a second        DNA-binding polypeptide, wherein said first DNA-binding        polypeptide is a DNA-binding polypeptide of the application and        wherein said second DNA-binding polypeptide is a DNA-binding        polypeptide of the application; or    -   a set comprising a first DNA-binding polypeptide and a second        DNA-binding polypeptide, wherein said first DNA-binding        polypeptide is a DNA-binding polypeptide of the application and        wherein said second DNA-binding polypeptide is not a DNA-binding        polypeptide of the application (said set may herein after be        more particularly referred to as “a mixed polypeptide set of the        application”).

The phrase “set” is intended in accordance with its ordinary meaning inthe field. It notably encompasses the meaning of “a plurality of”, moreparticularly the meaning of “a pair of”. Said set of plurality (or pair)can e.g., be in the form of one composition or kit, or of at least twocompositions or at least two kits.

Said one composition or kit comprises both said first and secondDNA-binding polypeptides.

Said at least two compositions or kits are in the form of separatecompositions or kits, each comprising one of said first and secondDNA-binding polypeptides (e.g., a first composition or kit comprisingsaid first DNA-binding polypeptide and a second composition or kitcomprising said second DNA-binding polypeptide, wherein said firstcomposition or kit is distinct or separate from said second compositionor kit). Said at least two compositions or kits can be for simultaneous,separate, distinct or sequential use, more particularly for simultaneousor sequential use.

In said polypeptide set, the first and second DNA-binding polypeptidescan e.g., be present as isolated polypeptides, as individualpolypeptides, as dimerized polypeptides, or can be contained withincell(s), e.g., within host and/or genetically engineered cell(s) (e.g.,as described below) (the first DNA-binding polypeptide can be containedwithin the same cell as said second DNA-binding polypeptide, or in twodistinct cells respectively).

According to an aspect of the application, the first DNA-bindingpolypeptide and the second DNA-binding polypeptide, which are comprisedin said set, are different from each other.

According to an aspect of the application, said first and secondDNA-binding polypeptides (specifically) bind to the same DNA nucleicacid but at different DNA target sites.

More particularly, said first and second DNA-binding polypeptides(specifically) bind to the same double-stranded DNA nucleic acid,wherein said first DNA-binding polypeptide binds to one strand of saiddouble-stranded DNA nucleic acid, and wherein said second DNA-bindingpolypeptide binds to the other strand of said double-stranded DNAnucleic acid (i.e., to the complementary strand). Hence, said firstDNA-polypeptide recognizes or binds to a first DNA target site, saidsecond DNA-polypeptide recognizes or binds to a second DNA target site,wherein said first DNA target site is comprised in a strand of adouble-stranded nucleic acid and said second DNA target site iscomprised in the other (complementary) strand of the samedouble-stranded DNA nucleic acid.

Advantageously, said first DNA target site is different from said secondDNA target site.

Advantageously, said first DNA target site is comprised in a firststrand of a double-stranded DNA nucleic acid, without being comprised inthe second strand of the same double-stranded DNA nucleic acid, and,conversely, said second DNA target site is comprised in said secondstrand (of the same double-stranded DNA nucleic acid as said first DNAtarget site), without being comprised in said first strand.

The application thus relates to a composition or kit comprising a firstDNA-binding polypeptide and a second DNA-binding polypeptide,

wherein said first DNA-binding polypeptide is different from said secondDNA-binding polypeptide,wherein each of said first and second DNA-binding polypeptides binds toa DNA nucleic acid comprising at least one DNA tandem repeat, whereinthe DNA nucleic acid to which said first DNA-binding polypeptide bindsis one strand of a double-stranded nucleic acid, wherein the DNA nucleicacid to which said second DNA-binding polypeptide binds is the otherstrand of the same double-stranded nucleic acid,wherein said double-stranded DNA nucleic acid is a gene involved in aneurological and/or muscular and/or skeletal disorder or diseaseinvolving said at least one DNA tandem repeat, wherein each of saidfirst and second DNA-binding polypeptides comprises a TAL effectortandem repeat consisting of adjacent units of TAL effector tandemrepeat,wherein the ordered series of RVDs formed by the RVDs respectivelycontained in said adjacent units of TAL effector tandem repeat, in N- toC-orientation, is an ordered series of amino acids, which determines therecognition of the 5′-3′ nucleotide sequence of a DNA target sitecontained in the strand of double-stranded DNA nucleic acid to whichsaid DNA-binding polypeptide binds,wherein the sequence of said DNA target site is:

-   -   i. a fragment of said strand of double-stranded DNA nucleic acid        consisting of a fragment of said at least one DNA tandem repeat,        wherein said fragment comprises more than one copy of said DNA        sequence unit of said at least one DNA tandem repeat, or    -   ii. a fragment of said strand of double-stranded DNA nucleic        acid, which starts outside the sequence of said at least one DNA        tandem repeat and ends within the sequence of said at least one        DNA tandem repeat, or conversely, which starts within the        sequence of said at least one DNA tandem repeat and ends outside        the sequence of said at least one DNA tandem repeat, wherein        each of said first and second DNA-binding polypeptides is        directly or indirectly linked to one endonuclease monomer or to        one fragment of endonuclease monomer, wherein said fragment of        endonuclease monomer still comprises the catalytic domain of        said endonuclease monomer, and        wherein said first and second DNA-binding polypeptides induce a        partial or complete deletion of said at least one DNA tandem        repeat.

Advantageously, said endonuclease monomer is the monomer of a dimericendonuclease.

According to an aspect of the application, the DNA target site is anon-overlapping DNA target site (as defined above) for only one of saidfirst and second DNA-binding polypeptides, or for each of said first andsecond DNA-binding polypeptides. When the DNA target site is anon-overlapping DNA target site (as defined above) for only one of saidfirst and second DNA-binding polypeptides, the DNA target site of theother of said first and second DNA-binding polypeptides is:

-   -   an overlapping DNA target site (as above-defined), or is    -   a DNA target site, which is neither a non-overlapping site (as        above-defined) nor an overlapping site (as above-defined).

According to an aspect of the application, the DNA target site is anoverlapping DNA target site (as defined above) for only one of saidfirst and second DNA-binding polypeptides, or for each of said first andsecond DNA-binding polypeptides. When the DNA target site is anoverlapping DNA target site (as defined above) for only one of saidfirst and second DNA-binding polypeptides, the DNA target site of theother of said first and second DNA-binding polypeptides is:

-   -   a non-overlapping DNA target site (as above-defined), or is    -   a DNA target site, which is neither a non-overlapping site (as        above-defined) nor an overlapping site (as above-defined).

Advantageously, the DNA target site is an overlapping DNA target site(as defined above) for one of said first and second DNA-bindingpolypeptides.

Advantageously, the DNA target site is a non-overlapping DNA target site(as defined above) for one of said first and second DNA-bindingpolypeptides.

Advantageously, the DNA target site is an overlapping DNA target site(as defined above) for one of said first and second DNA-bindingpolypeptides and is a non-overlapping DNA target site (as defined above)for the other of said first and second DNA-binding polypeptides.

This configuration drastically reduces the chance that the first andsecond DNA-binding polypeptides induce a length alteration or mutationat an off-target location, e.g., in a non-pathological gene, which wouldcomprise the same DNA repeat unit as the targeted pathological gene.

For example, said first DNA-binding polypeptide binds to a DNA targetsite of SEQ ID NO: 10 and said second DNA-binding polypeptide binds to aDNA target site of SEQ ID NO: 4, or said first DNA-binding polypeptidebinds to a DNA target site of SEQ ID NO: 11 and said second DNA-bindingpolypeptide binds to a DNA target site of SEQ ID NO: 5 (cf. FIG. 1B).

For example, the ordered series of RVDs formed by the adjacent unitsforming the TAL effector tandem repeat of said first DNA-bindingpolypeptide is NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD;NG, which determines the (specific) recognition (and the (specific)binding to) the non-overlapping DNA target site of SEQ ID NO: 10), andthe ordered series of RVDs formed by the adjacent units forming the TALeffector tandem repeat of said second DNA-binding polypeptide is NN; NG;NN; NI; NG; HD; HD; HD; HD; HD; HD; NI; NN; HD; NI, which determines the(specific) recognition (and the (specific) binding to) the overlappingDNA target site of SEQ ID NO: 4.

More particularly, the TAL effector tandem repeat of said firstDNA-binding polypeptide is different from the one of said secondDNA-binding polypeptide. The difference can be a difference in aminoacid sequence and/or in amino acid length.

More particularly:

-   -   the frame sequence(s) of the TAL effector tandem repeat units of        said first DNA-binding polypeptide is(are) different from the        one(s) of said second DNA-binding polypeptide; and/or    -   said first DNA-binding polypeptide and said second DNA-binding        polypeptide have different DNA target sites, i.e., the ordered        series of RVDs formed by the units of the TAL effector tandem        repeat that is contained in said first DNA-binding polypeptide        is different from the ordered series of RVDs formed by the units        of the TAL effector tandem repeat that is contained in said        second DNA-binding polypeptide.

For example:

-   -   the adjacent units of the TAL effector tandem repeat that is        contained in said first DNA-binding polypeptide may comprise one        or several copy(ies) of at least one sequence selected from the        group consisting of SEQ ID NOs: 25, 26, 46, 55 and said variants        thereof,    -   the adjacent units of the TAL effector tandem repeat that is        contained in said second DNA-binding polypeptide may comprise        one or several copy(ies) of at least one sequence selected from        the group consisting of SEQ ID NOs: 25, 26, 46, 55 and said        variants thereof, and    -   the ordered series of RVDs formed by the RVDs respectively        contained in the adjacent units of the TAL effector tandem        repeat of said first DNA-binding polypeptide, in N- to        C-orientation, is different from the ordered series of RVDs        formed by the RVDs respectively contained in the adjacent units        of the TAL effector tandem repeat of said second DNA-binding        polypeptide, in N- to C-orientation.

For example, the ordered series of RVDs formed by the RVDs respectivelycontained in the adjacent units of the TAL effector tandem repeat ofsaid first DNA-binding polypeptide, in N- to C-orientation, determinesthe recognition of (and the (specific) binding to) an overlapping DNAtarget site (as defined above, e.g., the DNA target site of SEQ ID NO: 4or 5, more particularly the DNA target site of SEQ ID NO: 4), and theordered series of RVDs formed by the RVDs respectively contained in theadjacent units of the TAL effector tandem repeat of said secondDNA-binding polypeptide, in N- to C-orientation, determines therecognition of (and the (specific) binding to) a non-overlapping DNAtarget site (as defined above, e.g., the DNA target site of SEQ ID NO:10 or 11, more particularly the DNA target site of SEQ ID NO: 10; cf.FIG. 1B).

Each of said first and second DNA-binding polypeptides can be linked toan endonuclease monomer or to a fragment of such a monomer as describedabove.

Advantageously, each of said first and second DNA-binding polypeptidesis linked to the monomer of a dimeric endonuclease, such as Fok I, or toa fragment of such a monomer as described above (cf. above).

In a polypeptide set of the application, said first DNA-bindingpolypeptide can be dimerized to said second DNA-binding polypeptide.

The application thus relates to a polymer, more particularly a dimer,which comprises said first and second DNA-binding polypeptides. Saidpolymer may further comprise at least one (double-stranded) DNA nucleicacid, more particularly at least one (double-stranded) DNA nucleic acidcomprising at least one DNA tandem repeat (as defined above). Said atleast one DNA nucleic acid can be linked to said first and secondDNA-binding polypeptides by non-covalent linkage, e.g., by non-covalentbinding of the RVDs of said first and second DNA-binding polypeptides tonucleotides of said at least one DNA nucleic acid, e.g., by non-covalentbinding of the RVDs of said first DNA-binding polypeptide to nucleotidesof one strand of said at least one double-stranded DNA nucleic acid andby non-covalent binding of the RVDs of said second DNA-bindingpolypeptide to nucleotides of the other (complementary) strand of thesame double-stranded DNA nucleic acid.

Alternatively, in a polypeptide set of the application, said firstDNA-binding polypeptide can be not dimerized to said second DNA-bindingpolypeptide.

More particularly, in a set of the application, said first DNA-bindingpolypeptide can be contained separately from said second DNA-bindingpolypeptide, e.g., to avoid dimerization of said first DNA-bindingpolypeptide to said second DNA-binding polypeptide.

The nucleotide length that extends from the DNA target site of saidfirst DNA-binding polypeptide to the DNA target site of said secondDNA-polypeptide is being referred to as the “spacer length”. Thisterminology is in accordance with the terminology that is used in thefield of TALENs.

Said spacer length is the number of nucleotides extending between thetwo proximal ends of the respective DNA target sites of said first andsecond DNA-binding polypeptides.

On a double-stranded DNA nucleic acid, wherein a first DNA-bindingpolypeptide recognizes or binds to a first DNA target site on one strandof said double-stranded DNA nucleic acid and wherein a secondDNA-binding polypeptide recognizes or binds to a second DNA target siteon the other strand of said double-stranded DNA nucleic acid, saidspacer length can be viewed as the nucleotide length that extends fromthe 3′ end of one of the respective DNA target sites of said first andsecond DNA-binding polypeptides to the 3′ end of the other of said DNAtarget sites (the last 3′ end nucleotides of said first and second DNAtarget sites are not taken into account in the computation of saidnucleotide number).

For example, in FIG. 1B, the sequence of the spacer isGCAGCAGCAGCAGCAGCAGC [SEQ ID NO: 8] (GCTGCTGCTGCTGCTGCTGC [SEQ ID NO: 9]on the complementary strand (5′-3′)). Hence, in FIG. 1B, the spacerlength is 20 nucleotides.

When each of the first and second DNA-binding polypeptides arerespectively linked to the monomer of the same dimeric endonuclease,said spacer length is selected to be sufficiently short and sufficientlylong for the two monomers of said dimeric endonuclease to dimerize whensaid first and second DNA-binding polypeptides are bound to theirrespective DNA target sites on each strand of the same double-strandedDNA nucleic acid (cf. FIGS. 1A and 1B). In other words, the respectiveDNA target sites of said first and second DNA-binding polypeptides areselected to be spaced apart by a spacer length that is appropriate fordimerization of the two endonuclease monomers respectively borne by saidfirst and second DNA-binding polypeptides.

The respective DNA target sites of said first and secondDNA-polypeptides can be spaced apart by a nucleotide length that mayvary from 6 to 40 nt (or bp), optimal cleavage being usually observedwith a spacer length of 10 to 30 nt (or bp), e.g., of 15-24 nt (or bp),15-21 nt (or bp) or 16-21 nt (or bp), e.g., 16, 17, 18, 19, 20 or 21 nt(or bp).

Advantageously:

-   -   said DNA target site is an overlapping DNA target site (as        defined above) for one of said first and second DNA-binding        polypeptides, and is a non-overlapping DNA target site (as        defined above) for the other of said first and second        DNA-binding polypeptides, and    -   each of said first and second DNA-binding polypeptides is linked        to the monomer of a dimeric endonuclease (cf. above), such as        Fok I, or to a fragment of such a monomer as described above.

Advantageously:

-   -   said DNA target site is an overlapping DNA target site (as        defined above) for one of said first and second DNA-binding        polypeptides, and is a non-overlapping DNA target site (as        defined above) for the other of said first and second        DNA-binding polypeptides,    -   each of said first and second DNA-binding polypeptides is linked        to the monomer of a dimeric endonuclease (cf. above), such as        Fok I, or to a fragment of such a monomer as described above,        and    -   the DNA target site of said first DNA-binding polypeptide is        spaced apart from the one of said second DNA-binding polypeptide        by a spacer length that enables dimerization of the two        endonuclease monomers respectively borne by said first and        second DNA-binding polypeptides (when said first and second        DNA-binding polypeptides are bound to their respective DNA        target sites), e.g., by a spacer length as indicated above e.g.,        a spacer length of 15-24 nt (or bp), 15-21 nt (or bp) or 16-21        nt (or bp), e.g., 16, 17, 18, 19, 20 or 21 nt (or bp).

In a set, which comprises a first DNA-binding polypeptide, which is aDNA-binding polypeptide of the application and which further comprises asecond DNA-binding polypeptide, which is not a DNA-binding polypeptideof the application, i.e., in a mixed polypeptide set of the application,said second DNA-binding polypeptide, which is not of the application,can e.g., be as above-defined except that its DNA target site is neithera non-overlapping DNA target site as defined above nor an overlappingDNA target site as defined above.

Said second DNA-binding polypeptide, which is not of the application,can e.g., be identical to a DNA-polypeptide of the application in allfeatures (e.g., it comprises a TAL effector tandem repeat asabove-defined), except for the DNA target site, which is not one that isrecognized by a DNA-binding polypeptide of the application.

Hence, said second DNA-binding polypeptide, which is not of theapplication, can e.g., be identical to a DNA-polypeptide of theapplication in all features (e.g., it comprises a TAL effector tandemrepeat as above-defined), except that the ordered series of RVDs formedby the RVDs respectively contained in the adjacent units of its TALeffector tandem repeat, in N- to C-orientation, is an ordered series ofamino acids, which according to the RVD/nucleotide correspondence shownin Table 5 above, determines the recognition of the 5′-3′ nucleotidesequence of a DNA target site that is contained in a DNA nucleic acid,wherein said DNA nucleic acid is as above-defined, but wherein said DNAtarget site is neither a non-overlapping DNA target site as definedabove nor an overlapping DNA target site as defined above. Moreparticularly, the DNA target site of said second DNA-bindingpolypeptide, which is not of the application, can be a fragment of saidDNA nucleic, which does not comprise any fragment of said at least oneDNA tandem repeat, more particularly a fragment of said DNA nucleic,which does not comprise any DNA sequence unit of said at least one DNAtandem repeat.

Said first DNA-binding polypeptide, which is comprised in the set withsaid second DNA-binding polypeptide, is a DNA-binding polypeptide of theapplication, and therefore has a DNA target site, which is either anon-overlapping DNA target site as above-defined or an overlapping DNAbinding site as above defined.

The spacer length is as above-defined. More particularly, the spacerlength between said first DNA-binding polypeptide of the application andsaid second DNA-binding polypeptide (which is not of the application) isa nucleotide length appropriate for dimerization of the two endonucleasemonomers respectively borne by said first and second DNA-bindingpolypeptides of the application. The respective DNA target sites of saidfirst and second DNA-polypeptides can be spaced apart by a nucleotidelength that may vary from 6 to 40 nt (or bp), optimal cleavage beingusually observed with a spacer length of 10 to 30 nt (or bp), e.g., of15-24 nt (or bp), 15-21 nt (or bp) or 16-21 nt (or bp), e.g., 16, 17,18, 19, 20 or 21 nt (or bp).

According to an aspect of the application, said first and secondDNA-binding polypeptides induce a double-strand break in saiddouble-stranded DNA nucleic acid. More particularly, they induce adouble-strand break specifically in said double-stranded DNA nucleicacid.

The application also relates to a nucleic acid, more particularly a DNAor RNA, more particularly a DNA. Said nucleic acid can be a man-made orartificial or engineered nucleic acid.

A nucleic acid of the application codes for the DNA-binding polypeptideof the application, more particularly for the DNA-binding polypeptide ofthe application (directly or indirectly) linked to (at least) oneendonuclease monomer (cf. above) or to (at least) one fragment ofendonuclease monomer as above-defined.

The application relates more particularly to a coding nucleic acid, thecoding sequence of which consists of a sequence coding for theDNA-binding polypeptide of the application, more particularly for theDNA-binding polypeptide of the application (directly or indirectly)linked to (at least) one endonuclease monomer (cf. above) or to (atleast) one fragment of endonuclease monomer as above-defined (saidcoding being according to the universal genetic code, taking due accountof its degeneracy).

The application relates more particularly to a coding nucleic acid, thecoding sequence of which comprises a sequence, which codes for the TALeffector tandem repeat of a DNA-binding polypeptide of the application(said coding being according to the universal genetic code, taking dueaccount of its degeneracy). Said coding sequence may e.g., comprise oneor several copy(ies) of at least one of the sequences coding for SEQ IDNO: 25, 26, 46, 55 and said variant sequences thereof.

Examples of such coding nucleic acid sequences comprise:

-   -   the nucleic acid sequence of SEQ ID NO: 45, which consists of 10        copies of a sequence coding for SEQ ID NO: 46 and of 5 copies of        a sequence coding for SEQ ID NO: 25, and which codes for a TAL        effector tandem repeat, wherein the ordered series of RVDs        (i.e., NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD;        NG) determine the (specific) recognition of (and the (specific)        binding to) the non-overlapping DNA target site of SEQ ID NO:        10),    -   the portion of the nucleic acid sequence of the insert carried        by plasmid pCLS9996exp (C.N.C.M. deposit number I-4804), which        codes for a TAL effector tandem repeat (said TAL effector tandem        repeat (specifically) binds to the non-overlapping DNA target        site of SEQ ID NO: 10),    -   the nucleic acid sequence of SEQ ID NO: 54, which consists of 5        copies of a sequence coding for SEQ ID NO: 46, 2 copies of a        sequence coding for SEQ ID NO: 55 and of 8 copies of a sequence        coding for SEQ ID NO: 25, and which codes for a TAL effector        tandem repeat, wherein the ordered series of RVDs (i.e., NN; NG;        NN; NI; NG; HD; HD; HD; HD; HD; HD; NI; NN; HD; NI) determine        the (specific) recognition of (and the (specific) binding to)        the overlapping DNA target site of SEQ ID NO: 4),    -   the portion of the nucleic acid sequence of the insert carried        by plasmid pCLS16715 (C.N.C.M. deposit number I-4805), which        codes for a TAL effector tandem repeat (said TAL effector tandem        repeat (specifically) binds to the overlapping DNA target site        of SEQ ID NO: 4).

The application more particularly relates to a nucleic acid (DNA orRNA), which codes for the TAL effector tandem repeat coded by plasmidpCLS9996exp (C.N.C.M. deposit number I-4804) or by plasmid pCLS16715(C.N.C.M. 1-4805).

Examples of sequences coding for an endonuclease monomer or for afragment of endonuclease monomer as above-defined comprise:

-   -   the sequence of SEQ ID NO: 3 (which codes for the FokI monomer        of SEQ ID NO: 49) and the fragments thereof, which still code        for the FokI catalytic domain, and    -   the sequences coding for the I-TevI endonuclease and the        fragments thereof, which still code for the I-TevI catalytic        domain.

The nucleic acid of the application may comprise at least one codingsequence, wherein said at least one coding sequence codes for (at least)one DNA-binding polypeptide of the application (directly or indirectly)linked to (at least) one endonuclease monomer (cf. above) or to (atleast) one fragment of endonuclease monomer as above-defined (accordingto the universal genetic code and taking due account of its degeneracy).Examples of such coding nucleic acid sequences comprise:

-   -   the nucleic acid sequence of SEQ ID NO: 39 (which codes for a        TALEN, which (specifically) binds to the non-overlapping DNA        target site of SEQ ID NO: 10),    -   the nucleic acid sequence of the insert carried by plasmid        pCLS9996exp (C.N.C.M. deposit number I-4804), which codes for a        TALEN that (specifically) binds to the non-overlapping DNA        target site of SEQ ID NO: 10),    -   the nucleic acid sequence of SEQ ID NO: 50 (which codes for a        TALEN, which (specifically) binds to the overlapping DNA target        site of SEQ ID NO: 4),    -   the nucleic acid sequence of the insert carried by plasmid        pCLS16715 (C.N.C.M. deposit number I-4805), which codes for a        TALEN that (specifically) binds to the overlapping DNA target        site of SEQ ID NO: 4).

The nucleic acid of the application can further comprise a translationalstart codon, such as ATG, located (immediately) in 5′ of said codingsequence and/or further comprise a 3′UTR for transcription terminationand polyadenylation of RNA transcript located (immediately) in 3′ ofsaid coding sequence. For example, said 3′ UTR comprises a translationalstop codon (such as TGA, TAG or TAA) and a polyA sequence.

The nucleic acid of the application may further comprise sequence(s),which does(do) not code for amino acid(s) but which regulates(regulate)transcription and/or translation. For example, the nucleic acid of theapplication can further comprise (at least) one sequence for initiatingDNA transcription located in 5′ of said coding sequence and/or furthercomprise (at least) one sequence for terminating DNA transcriptionlocated in 3′ of said coding sequence. For example, the nucleic acid ofthe application may further comprise (at least) one enhancer (such asthe GAL10 enhancer of SEQ ID NO: 37) and a promoter (such as the CYC1promoter of SEQ ID NO: 38) in 5′ of said coding sequence, and mayfurther comprise a terminator (such as an ADH1 terminator of SEQ ID NO:40 or 51).

The nucleic acid of the application, can (thereby) form an expressioncassette for expression in a host cell, more particularly in aeukaryotic host cell, more particularly in a mammalian cell, a non-humanmammalian cell (e.g., a rodent cell, such as a mouse cell), a human hostcell, a yeast host cell, a bacterial host cell or a plant host cell,more particularly in a human host cell or a yeast host cell, moreparticularly in a human host cell.

Hence, the nucleic acid of the application may consist of:

-   -   (at least) one sequence coding for a polypeptide consisting of        the DNA-binding polypeptide of the application (according to the        universal genetic code and taking due account of its        degeneracy), or coding for a polypeptide consisting of the        DNA-binding polypeptide of the application (directly or        indirectly) linked to (at least) one endonuclease monomer (cf.        above) or to (at least) one fragment of endonuclease monomer as        above-defined (according to the universal genetic code and        taking due account of its degeneracy), and    -   optionally, sequence(s), which does(do) not code for amino acids        but which regulates(regulate) transcription and/or translation,        such as (at least) one sequence for initiating DNA transcription        located (immediately) in 5′ of said nucleic acid sequence and/or        (at least) one sequence for terminating DNA transcription        located (immediately) in 3′ of said nucleic acid sequence, e.g.,        as above described.

For example, the sequence of the nucleic acid of the application cancomprise or consist of the sequence of SEQ ID NO: 2 or the sequence ofSEQ ID NO: 1.

The sequence of SEQ ID NO: 2 codes for a DNA-binding polypeptide of theapplication, which is linked to a FokI endonuclease monomer and, which(specifically) binds to a DNA target site that is the (non-split) leftTALE DNA-binding domain of FIG. 1B (cf. example 1 below). The sequenceof the left-hand TALE DNA-binding domain of FIG. 1B is the sequence ofSEQ ID NO: 4, i.e., an overlapping DNA target site as defined above.

The sequence of SEQ ID NO: 1 codes for a DNA-binding polypeptide of theapplication, which is linked to a FokI endonuclease monomer and, which(specifically) binds to a DNA target site that is the right TALEDNA-binding domain of FIG. 1B (cf. example 1 below). The sequence of theDNA target site of the right TALE DNA-binding domain of FIG. 1B is thesequence of SEQ ID NO: 10, i.e., a non-overlapping DNA target site asdefined above.

For example, the sequence of the nucleic acid of the application cancomprise or consist of the sequence of the insert carried by plasmidpCLS9996 (C.N.C.M. I-4804) or the sequence of the insert carried byplasmid pCLS16715 (C.N.C.M. I-4805).

The application also relates to a nucleic acid vector, more particularlya recombinant vector, more particularly a recombinant expression nucleicacid vector, which comprises at least one nucleic acid (DNA or RNA) ofthe application.

A nucleic acid vector of the application may comprise a cloning siteinto which a nucleic acid is inserted, wherein the sequence of saidinserted nucleic acid is the sequence of the nucleic acid of theapplication.

The nucleic acid vector of the application advantageously is anon-integrative (i.e., a vector, which does not induce the integrationof the nucleic acid into the genome of the host into which said vectorhas been introduced) and/or non-replicative.

According to an embodiment of the application, said nucleic acid vectoris a recombinant expression vector. More particularly, said nucleic acidvector is an expression vector comprising a cloning site into which anucleic acid to be expressed is inserted under the control of a 5′expression promoter (said promoter being inducible or non-inducible),and optionally under the control of at least one 5′ expression enhancer,wherein the sequence of said nucleic acid to be expressed is thesequence of the nucleic acid of the application.

Advantageously, said expression vector is a non-integrative vector, moreparticularly a vector for transient expression, for example a plasmid.

An illustrative plasmid is the plasmid pCLS16715 (C.N.C.M. I-4805),which carries the sequence of SEQ ID NO: 2 (as nucleic acid to beexpressed): SEQ ID NO: 2 codes for the TALEN monomer that binds to theleft-hand DNA target site of SEQ ID NO: 4; cf. FIG. 1B. Anotherillustrative plasmid is the plasmid pCLS9996exp (C.N.C.M. I-4804), whichcarries the sequence of SEQ ID NO: 1 (as nucleic acid to be expressed):SEQ ID NO: 1 codes for the TALEN monomer that binds to the right-handDNA target site of SEQ ID NO: 10; cf. FIG. 1B.

Each of plasmid pCLS16715 and plasmid pCLS9996exp has been deposited atthe Collection Nationale de Cultures de Microorganismes (C.N.C.M.) underthe terms of the Budapest Treaty (COLLECTION NATIONALE DE CULTURES DEMICROORGANISMES; Institut Pasteur; 28, rue du Docteur Roux; F-75724PARIS CEDEX 15; FRANCE).

The C.N.C.M. deposit number of plasmid pCLS16715 is I-4805 and the dateof the deposit under the terms of the Budapest Treaty is 10 Oct. 2013.Deposit I-4805 is plasmid pCLS16715 transformed in E. coli (moreparticularly, an E. coli strain, which is deficient in the genesinvolved in the rearrangement and deletion of DNA, such as E. coliSURE®2, which is available from STRATEGENE, an AGILENT TECHNOLOGIESdivision, California, U.S.A.; e.g, an E. coli strain, which is endA1glnV44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 uvrCe14-Δ(mcrCB-hsdSMR-mrr)171 F[proAB⁺ lacI^(q) lacZΔM15 Tn10 Amy Cm^(R)]).An example of suitable growth medium is Lysogeny Broth (LB) growthmedium+ampicillin (e.g., ampicillin at 100 μg/mL). An example ofsuitable incubation condition is 37° C. (more particularly, 37° C. understirring conditions).

The C.N.C.M. deposit number of plasmid pCLS9996exp is I-4804 and thedate of the deposit under the terms of the Budapest Treaty is 10 Oct.2013. Deposit I-4804 is plasmid pCLS9996 transformed in E. coli (moreparticularly, an E. coli strain, which is efficient in DNAtransformation and in maintenance of large plasmids, such as E. coliDH10B (cf. Durfee et al. 2008, J. Bacteriol. 190(7): 2597-2606)). Anexample of suitable growth medium is Lysogeny Broth (LB) growthmedium+kanamycin sulfate (e.g., kanamycin at 50 μg/mL). An example ofsuitable incubation condition is 37° C. (more particularly, 37° C. understirring conditions).

Appropriate non-integrative vectors, more particularly appropriatevectors for transient expression, also comprise retroviral or lentiviralvectors, more particularly HIV vectors, more particularly HIV1 vectors,wherein the integrase of said vectors is or has been made defective,e.g., by class 1 integrase mutation(s) (whereby said vectors are or havebeen made non-integrative). Examples of such non-integrative vectorscomprise:

-   -   a HIV1 vector, the integrase of which has been made defective by        replacement of the ²⁶²RRK motif by AAH as described in Philippe        et al. 2006 (cf. FIG. 1 of Philippe et al. 2006),    -   a retroviral or lentiviral vector, more particularly a HIV        vector, more particularly a HIV1 vector, as described in WO        99/55892, which has been made non-integrative e.g., by the        method described in Philippe et al. 2006 or by the method        described in WO 2006/010834,    -   a non-integrative vector as described in WO 2009/019612, more        particularly at paragraph

of WO 2009/019612.

The application more particularly relates to a recombinant vector, moreparticularly a recombinant expression vector, more particularly arecombinant retroviral expression vector, more particularly a lentiviralexpression vector, which comprises:

at least one nucleic acid (DNA or RNA) of the application, moreparticularly at least one RNA of the application (and regulatoryelements for the expression of said at least one nucleic acid or RNA),anda defective integrase, more particularly an integrase, which has beenmade defective by mutation(s), more particularly an integrase, which hasbeen made defective by mutation(s), wherein said mutation(s) comprise(s)or consist(s) of one or more point mutations affecting a basic region ofits C-terminal region.

Said defective integrase does not allow (or prevents) the integration ofsaid at least one nucleic acid or of the cDNA thereof into the genome ofa host cell, more particularly into the genome of a mammalian cell (moreparticularly into the genome of a mammalian neuronal cell and/or of amammalian muscular cell and/or of a cell of the mammalian skeleton),more particularly into the genome of a human cell (more particularlyinto the genome of a human neuronal cell and/or of a human muscular celland/or of a cell of the human skeleton).

Said defective integrase may e.g., be the integrase of HumanImmunodeficiency Virus type 1 (HIV1), Human Immunodeficiency Virus type2 (HIV2), Simian Immunodeficiency Virus (SIV), Feline ImmunodeficiencyVirus (FIV), Equine Infectious Anemia Virus (EIAV), BovineImmunodeficiency Virus (BIV), visna virus or Caprine ArthritisEncephalitis Virus (CAEV), more particularly the integrase of HIV1,which has been made defective by mutation(s), more particularly anintegrase of HIV1, which has been made defective by mutation(s), whereinsaid mutation(s) comprise(s) or consist(s) of one or more pointmutations affecting a basic region of its C-terminal region (cf. WO2006/010834). More particularly, said integrase may e.g., be theintegrase of HIV1, which has been made defective by replacement of the²⁶²RRK motif by AAH (cf. Philippe et al. 2006).

Said recombinant vector, more particularly a recombinant expressionvector, more particularly a recombinant retroviral expression vector,more particularly a lentiviral expression vector advantageously isnon-integrative, more particularly non-integrative and non-replicative.

The application more particularly relates to a recombinant retroviralexpression vector, more particularly a lentiviral expression vector,more particularly a HIV expression vector, more particularly a HIV1expression vector, which comprises at least one nucleic acid or RNA ofthe application (and regulatory elements for the expression of said atleast one nucleic acid), wherein the integrase of said retrovirus orlentivirus has been made defective, more particularly which has beenmade defective by mutation(s), more particularly which has been madedefective by mutation(s), wherein said mutation(s) comprise(s) orconsist(s) of one or more point mutations affecting a basic region ofits C-terminal region (cf. WO 2006/010834). Said retrovirus orlentivirus may e.g., be HIV1, HIV2, SIV, FIV, EIAV, BIV, visna or CAEV,more particularly HIV1.

The application more particularly relates to a recombinant HIV1expression vector, which comprises at least one nucleic acid or RNA ofthe application (and regulatory elements for the expression of said atleast one nucleic acid), wherein the integrase of said HIV1 has beenmade defective, more particularly which has been made defective bymutation(s), more particularly which has been made defective bymutation(s), wherein said mutation(s) comprise(s) or consist(s) of theeplacement of the ²⁶²RRK motif by AAH (cf. Philippe et al. 2006).

Said recombinant vector, more particularly said recombinant expressionvector, more particularly said recombinant retroviral expression vector,more particularly said lentiviral expression vector, more particularlysaid HIV1 vector, may further comprise a recombinant genome, which isdevoid of, or has been deleted from, all the lentiviral encodingsequences, and which comprises, between the lentiviral LTR 5′ and 3′sequences, a lentiviral encapsidation psi sequence, a RNA nuclear exportelement, a transgene comprising said at least one nucleic acid, andoptionally, a promoter and/or a sequence favoring the nuclear import ofRNA (cf. WO 99/55892).

Appropriate vectors comprise vectors, which are especially adapted forthe expression of the nucleic acid of the application by, or in, aparticular type of cells, tissue(s) or organ(s), for example, vectors,which are especially adapted for the expression of the inserted nucleicacid by neuronal cells, more particularly:

-   -   an expression lentivirus-derived vector, more particularly, a        non-replicative expression lentivirus-derived vector (e.g., as        described in WO 2013/068430, cf. pages 35-44 of WO 2013/068430        and the examples of WO 2013/068430) or    -   a lentiviral vector pseudotyped particle, more particularly a        lentiviral vector, which has been pseudotyped with the G protein        of a rabies virus (e.g., as described in WO 2013/068430, cf.        pages 41-44 of WO 2013/068430 and the examples of WO        2013/068430).

The application more particularly relates to a recombinant vector, moreparticularly a recombinant expression vector, more particularly arecombinant (expression) plasmid, which comprises:

i. at least one nucleic acid of the application, more particularly atleast one RNA of the application,ii. expression regulatory elements of said at least one nucleic acid orRNA,iii. a cis-acting central initiation region (cPPT) and a cis-actingtermination region (CTS), both of lentiviral origin, andiv. regulatory signals of retroviral origin (more particularly, oflentiviral origin) for transcription (more particularly, for reversetranscription), expression and packaging.

Examples of the structure of such vectors (elements ii., iii. and iv.)are described in WO 2013/068430, more particularly from page 35 line 25to page 41 line 4.

Said recombinant vector, more particularly said recombinant expressionvector, more particularly said recombinant (expression) plasmidadvantageously is non-replicative. Non-replication may be achieved byany means that the person of ordinary skill in the art may findappropriate, e.g., by deletion and/or mutation(s) of viral sequence(s)(e.g., of the gag and/or pol and/or env gene(s)) and/or of cis-actinggenetic elements needed for particle formation (cf. WO 2013/068430, moreparticularly from page 40 line 26 to page 41 line 4).

The application also relates to a recombinant viral particle, moreparticularly to a lentiviral vector pseudotyped particle, comprising atleast one nucleic acid vector of the application.

The application also relates to a recombinant viral particle, moreparticularly to a lentiviral vector pseudotyped particle, comprising GAGstructural proteins and a viral core made of (a) POL proteins and (b) alentiviral genome comprising said at least one nucleic acid or RNA ofthe application, expression regulatory elements of said at least onenucleic acid or RNA, a cis-acting central initiation region (cPPT) and acis-acting termination region (CTS), both of lentiviral origin, andregulatory signals of retroviral origin for transcription (moreparticularly for reverse transcription), expression and packaging,wherein said particle is pseudotyped with the G protein of a VesicularStomatitis Indiana Virus (VSIV or VSV) or with the G protein of a rabiesvirus (cf. above and WO 2013/068430, more particularly from page 41 line6 to page 44 line 28). Said rabies virus can e.g., be the ERA strain(ATCC vr332) or the CVS strain (ATCC vr959). The sequence of the Gprotein of the ERA strain is available under accession number AF406693.The sequence of the G protein of the CVS rabies virus strain isavailable under accession number AF406694. Said recombinant viralparticle, more particularly said lentiviral vector pseudotyped particle,may advantageously have been made defective, i.e., the integrase oflentiviral origin (which is coded by the pol gene) is devoid of thecapacity of integration of the lentiviral genome into the genome of ahost cell, more particularly into the genome of a mammalian cell (moreparticularly into the genome of a mammalian neuronal cell and/or of amammalian muscular cell and/or of a cell of the mammalian skeleton),more particularly into the genome of a human cell (more particularlyinto the genome of a human neuronal cell and/or of a human muscular celland/or of a cell of the human skeleton). Said integrase may e.g.,comprise mutation(s), which alter(s) or impede(s) its integraseactivity. Examples of such defective integrases and of such mutation(s)are described in WO 2013/068430 from page 43 line 6 to page 44 line 28.

The application also relates to a set comprising a nucleic acid of theapplication and a nucleic acid vector of the application.

The application also relates to a set comprising a first nucleic acidand a second nucleic acid, wherein only one, or each one, of said firstand second nucleic acids is a nucleic acid of the application. Saidfirst nucleic acid is different from said second nucleic acid.

When only one of said first and second nucleic acids is a nucleic acidof the application, the other of said first and second nucleic acid is anucleic acid, which is not of the application. For example, the setcomprises a first nucleic acid, which is a nucleic acid of theapplication, and a second nucleic acid, which is not of the application,wherein said first nucleic acid of the application codes for a firstDNA-binding polypeptide and said second nucleic acid codes for a secondDNA-binding polypeptide, and wherein said first and second DNA-bindingpolypeptides are the first and second DNA-binding polypeptides of amixed polypeptide set of the application as defined above (firstDNA-binding polypeptide, which is of the application, and secondDNA-binding polypeptide, which is not of the application; cf. above).

The application more particularly relates to a set wherein each of saidfirst and second nucleic acids is a nucleic acid of the application.

The application also relates to a set comprising a first nucleic acidvector and a second nucleic acid vector, wherein only one, or each one,of said first and second nucleic acid vectors is a nucleic acid vectorof the application. Said first nucleic acid vector is different fromsaid second nucleic acid vector.

When only one of said first and second nucleic acid vectors is a nucleicacid vector of the application, the other of said first and secondnucleic acid vectors is a nucleic acid vector, which is not of theapplication. For example, the set comprises a first nucleic acid vector,which comprises a nucleic acid of the application, and a second nucleicacid vector, which comprises a nucleic acid, which is not of theapplication, wherein said first nucleic acid of the application codesfor a first DNA-binding polypeptide and said second nucleic acid codesfor a second DNA-binding polypeptide, and wherein said first and secondDNA-binding polypeptides are the first and second DNA-bindingpolypeptides of a mixed polypeptide set of the application as definedabove (first DNA-binding polypeptide, which is of the application, andsecond DNA-binding polypeptide, which is not of the application; cf.above).

The application more particularly relates to a set, wherein each of saidfirst and second nucleic acid vectors is a nucleic acid vector of theapplication.

Each of these sets can be herein referred to as the “nucleic acid/vectorset of the application”. The phrase “set” is intended in accordance withits ordinary meaning in the field. It notably encompasses the meaning of“a plurality of”, more particularly the meaning of “a pair of”. Said setof plurality can e.g., be in the form of one composition or kit, or ofat least two compositions or of at least two kits.

Said one composition or kit comprises both said first and secondDNA-binding nucleic acids or nucleic acid vectors.

Said at least two compositions or kits are in the form of separatecompositions or kits, each comprising one of said first and secondnucleic acids or nucleic acid vectors (e.g., a first composition or kitcomprising said first nucleic acid or nucleic acid vector and a secondcomposition or kit comprising said second nucleic acid or nucleic acidvector, wherein said first composition or kit is distinct or separatefrom said second composition or kit). Said at least two compositions orkits can be for simultaneous, separate, distinct or sequential use, moreparticularly for simultaneous or sequential use.

In said nucleic acid/vector set, the first and second nucleic acids orvectors can e.g., be present as isolated nucleic acids or vectors, asindividual nucleic acids or vectors, or can be contained within cell(s),e.g., host and/or genetically engineered cell(s) as described below (thefirst nucleic acid or vector can be contained within the same cell assaid second nucleic acid or vector, or in two distinct cellsrespectively).

The application relates to a composition or kit comprising:

a first recombinant nucleic acid vector and a second recombinant nucleicacid vector, wherein said first recombinant nucleic acid vector isdifferent from said second recombinant nucleic acid vector and whereinsaid first recombinant nucleic acid vector and said second recombinantnucleic acid vector respectively code for the first DNA-bindingpolypeptide and for the second DNA-binding polypeptide as defined above;and/or comprisinga first lentiviral vector pseudotyped particle and a second lentiviralvector pseudotyped particle, wherein said first lentiviral vectorpseudotyped particle is different from said second lentiviral vectorpseudotyped particle and wherein said first lentiviral vectorpseudotyped particle and said second lentiviral vector pseudotypedparticle respectively code for the first DNA-binding polypeptide and forthe second DNA-binding polypeptide as defined above.

More particularly, each of said first and second recombinant nucleicacid vectors is a recombinant nucleic acid vector of the application,and/or each of said first and second lentiviral vector pseudotypedparticles is a lentiviral vector pseudotyped particle of theapplication.

In said nucleic acid/vector set, said first nucleic acid and/or saidsecond nucleic acid can be contained in/on a nanoparticle or liposome asdescribed below, and/or said first nucleic acid vector and/or saidsecond nucleic acid vector can be contained in/on a nanoparticle orliposome as described below.

Said nucleic acid/vector set can be contained in a composition suitablefor nucleic acid transfection of a cell, more particularly of aeukaryotic cell, more particularly of a mammalian cell, a non-humanmammalian cell (e.g., a rodent cell, such as a mouse cell), a humancell, a yeast cell, a bacterial cell or a plant cell, more particularlyof a human cell or a yeast cell, more particularly of a human cell.

The term “transfection” herein encompasses its broadest general meaningin the field of genetic engineering. It notably encompasses any processof deliberately introducing a nucleic acid into a cell (said process canbe virus-mediated or not virus-mediated, said cell can be eukaryotic ornot eukaryotic).

Said nucleic acid/vector set may further comprises at least one cell,more particularly at least one eukaryotic cell, more particularly atleast one mammalian cell, at least one non-human mammalian cell (e.g., arodent cell, such as a mouse cell), at least one human cell, at leastone yeast cell, at least one bacterial cell or at least one plant cell,more particularly at least one human cell or at least one yeast cell,more particularly at least one human cell.

The application also relates to a nanoparticle or to a liposome, whichcomprises at least one of the polypeptides, sets, nucleic acids, vectorsand host cells of the application, more particularly which comprises atleast one of the polypeptides, sets and host cells of the application.Said at least one polypeptide, set, nucleic acid, vector or host cell ofthe application can be contained in and/or on nanoparticles. Said atleast one polypeptide, set, nucleic acid, vector or host cell of theapplication can be contained in and/or on a liposome, e.g., it can beencapsulated inside a liposome or associated to a liposome deliverysystem. Said liposome can e.g., be a cationic liposome, a pegylatedliposome. Said liposome can be loaded with nanoparticles. Thenanoparticle and/or liposome formulation of the polypeptide, set,nucleic acid, vector or host cell of the application is notably usefulfor improved crossing of the blood-brain barrier and/or for protectionagainst serum degradation.

The application also relates to a cell, more particularly a eukaryoticcell more particularly a mammalian cell, a non-human mammalian cell, ahuman cell, a yeast cell, a bacterial cell or a plant cell, moreparticularly a human cell or a yeast cell, more particularly a humancell, which comprises at least one DNA-binding polypeptide of theapplication and/or at least one polypeptide set of the applicationand/or at least one nucleic acid of any one of the application and/or atleast one nucleic acid vector of the application and/or at least onenucleic acid/vector set of the application and/or at least one liposomeor nanoparticle of the application.

Said cell can e.g., be a host cell and/or a recombinant cell and/or agenetically engineered cell. The application also relates to the invitro use of said cell for the production or synthesis of at least oneDNA-binding polypeptide of the application and/or at least onepolypeptide set of the application and/or at least one nucleic acid ofthe application and/or at least one nucleic acid vector of theapplication and/or at least one nucleic acid/vector set of theapplication and/or at least one liposome or nanoparticle of theapplication.

The application also relates to an in vitro method for the production ofa product, which binds, or specifically binds, to a (double-stranded)DNA nucleic acid comprising at least one DNA tandem repeat, moreparticularly which cleaves, or specifically cleaves, a (double-stranded)DNA nucleic acid comprising at least one DNA tandem repeat, moreparticularly which fully or partially deletes said at least one DNAtandem repeat, more particularly which fully or partially deletes saidat least one DNA tandem repeat in a specific manner.

Said method typically comprises in vitro growing said cell of theapplication on a culture medium, allowing it to produce said at leastone DNA-binding polypeptide of the application and/or at least onenucleic acid of the application and/or at least one nucleic acid vectorof the application, and collecting said at least one DNA-bindingpolypeptide of the application and/or at least one nucleic acid of theapplication and/or at least one nucleic acid vector of the application.

The application also relates to a method for producing (at least one)DNA-binding polypeptide, which binds, or specifically binds, to a DNAnucleic acid comprising at least one DNA tandem repeat, wherein saidmethod comprises producing or synthesizing a DNA-binding polypeptide ofthe application.

The application also relates to a method for producing a pair ofDNA-binding polypeptides, which comprises producing or synthesizing afirst DNA-binding polypeptide and a second DNA-binding polypeptide,wherein said first DNA-binding polypeptide and said second DNA-bindingpolypeptide are as defined above for a polypeptide set of theapplication.

The expression “synthesizing a polypeptide” encompasses synthesizing apolypeptide by chemical synthesis (e.g., by solid phase synthesis, or byliquid phase synthesis), as well as synthesizing a polypeptide byrecombinant expression. More particularly, said expression encompassesthe synthesis of a polypeptide by recombinant expression, moreparticularly by recombinant expression of a nucleic acid of theapplication and/or of a nucleic acid vector of the application and/orfrom a host cell of the application and/or from a composition comprisinga first nucleic acid and a second nucleic acid of the application (asdefined above). Said method may further comprise the collection of thesynthesized polypeptide, e.g., by purification and/or isolation, forexample by antibody capture and/or by HPLC.

The application also relates to a non-human animal (e.g., a rodent, suchas a mouse, or pig, or a rabbit), which has been engineered to containor produce at least one DNA-binding polypeptide of the applicationand/or at least one polypeptide set of the application and/or at leastone nucleic acid of the application and/or at least one nucleic acidvector of the application and/or at least one nucleic acid/vector set ofthe application and/or at least one cell of the application.

The application also relates to the use of said non-human animal for theproduction or synthesis of at least one DNA-binding polypeptide of theapplication and/or at least one nucleic acid of the application and/orat least one nucleic acid vector of the application.

The application also relates to a method for the production of aproduct, which binds, or specifically binds, to a (double-stranded) DNAnucleic acid comprising at least one DNA tandem repeat, moreparticularly which cleaves, or specifically cleaves, a (double-stranded)DNA nucleic acid comprising at least one DNA tandem repeat, moreparticularly which fully or partially deletes said at least one DNAtandem repeat, more particularly which fully or partially deletes saidat least one DNA tandem repeat in a specific manner.

Said method typically comprises breeding or keeping said non-humananimal, allowing it to produce at least one DNA-binding polypeptide ofthe application and/or at least one nucleic acid of the applicationand/or at least one nucleic acid vector of the application, andcollecting from said animal at least one DNA-binding polypeptide of theapplication and/or at least one nucleic acid of the application and/orat least one nucleic acid vector of the application.

The application also relates to a composition, more particularly to apharmaceutical composition, medicament, drug or kit, which comprises atleast one product of the application, more particularly at least oneDNA-binding polypeptide of the application and/or at least onepolypeptide set of the application and/or at least one nucleic acid ofthe application and/or at least one nucleic acid vector of theapplication and/or at least one nucleic acid/vector set of theapplication and/or at least one liposome or nanoparticle of theapplication and/or at least one cell of the application.

Said pharmaceutical composition, medicament, drug or kit may furthercomprise at least one pharmaceutically acceptable vehicle or carrier,more particularly a physiologically acceptable vehicle or carrier, moreparticularly a vehicle or carrier, which is adapted to the physiology ofa mammal, e.g., a human or non-human mammal. Said vehicle or carrier canbe mixed with said at least one product of the application.

Said vehicle or carrier can e.g., be or comprise one or several elementsselected from at least one diluent, at least one excipient, at least oneadditive, at least one pH adjuster, at least one pH buffering agent, atleast one emulsifier agent, at least one dispersing agent, at least onepreservative, at least one surfactant, at least one gelling agent, atleast one buffering agent, at least one stabilizing agent and at leastone solubilising agent.

Appropriate pharmaceutically acceptable vehicles and formulationsinclude all known pharmaceutically acceptable vehicles and formulations,such as those described in “Remington: The Science and Practice ofPharmacy”, 20^(th) edition, Mack Publishing Co.;

and “Pharmaceutical Dosage Forms and Drug Delivery Systems”, Ansel,Popovich and Allen Jr., Lippincott Williams and Wilkins.

When said composition, pharmaceutical composition, medicament, drug orkit is intended for administration to a subject (e.g., a non-humanmammal or a human) in need thereof, the nature of the vehicle will ingeneral depend on the particular mode of administration being employed.For instance, parenteral formulations usually comprise injectable fluidsthat include pharmaceutically and physiologically acceptable fluids,including water, physiological saline, balanced salt solutions, buffers,aqueous dextrose, glycerol, ethanol, sesame oil, combinations thereof,or the like as a vehicle. The medium may also contain conventionalpharmaceutical adjunct materials such as, for example, pharmaceuticallyacceptable salts to adjust the osmotic pressure, buffers, preservativesand the like.

In said composition, pharmaceutical composition, drug, medicament or kitof the application, said at least one product of the application cane.g., be formulated as, or contained in, a liquid solution, asuspension, an emulsion or a capsule. It can be formulated e.g., forimmediate release, or for differed release or sustained releaseformulation.

Advantageously, said composition, pharmaceutical composition, drug,medicament or kit is stored or contained in a sterile container and/orenvironment.

The application describes products, which are DNA-binding polypeptides,nucleic acids, sets, vectors, liposomes, nanoparticles, cells, non-humananimals, compositions, pharmaceutical compositions, medicaments, drugs,kits.

Each of these products is useful in the medical field, more particularlyin the field of the treatment and/or palliation and/or prevention of adisease or disorder.

Said disease or disorder can e.g., be any disorder or disease involvingat least one DNA tandem repeat (as above described), more particularlyat least one (direct) DNA tandem repeat in a DNA nucleic acid, moreparticularly at least one (direct) DNA tandem repeat in adouble-stranded DNA nucleic acid.

Said disease or disorder can e.g., be any disorder or disease involvingat least one expanded or abnormally-expanded DNA tandem repeat, moreparticularly at least one expanded or abnormally-expanded DNA tandemrepeat in a DNA nucleic acid, more particularly at least one expanded orabnormally-expanded DNA tandem repeat in a double-stranded DNA nucleicacid. The phrase “expanded” or “abnormally-expanded” means that thenumber of repeat units forming the DNA tandem repeat is above the normalaverage number (for the DNA nucleic acid in consideration).

Said disease or disorder can e.g., be a neurological and/or muscularand/or skeletal disorder or disease.

Said disease or disorder can e.g., be a neurological and/or muscularand/or skeletal disorder or disease involving at least one DNA tandemrepeat as above-described.

Said at least one DNA tandem repeat may e.g., have a non-linearsecondary structure such as a hairpin, a triple helix or a tetraplex.

Said disease or disorder can e.g., be a neurological and/or muscularand/or skeletal disorder or disease involving at least one DNA tandemrepeat in a double-stranded DNA nucleic acid, more particularly at leastone DNA tandem repeat in a double-stranded DNA nucleic acid, whereinsaid at least one DNA tandem repeat has a non-linear secondary structuresuch as a hairpin, a triple helix or a tetraplex.

Said at least one DNA tandem repeat can be contained in a gene, moreparticularly a eukaryotic gene, more particularly a non-mammalianeukaryotic gene, e.g., a yeast gene, more particularly a mammalian gene,e.g., a non-human mammalian gene or a human gene.

Advantageously, said at least one DNA tandem repeat is contained in achromosome, more particularly is a gene, a non-mammalian eukaryoticgene, a mammalian gene, a non-human mammalian gene or a human gene,wherein said gene is contained in a chromosome, more particularly ahuman chromosome.

Said at least one DNA tandem repeat can be contained in any location ofsaid gene, e.g., in a promoter and/or in the 5′UTR and/or in at leastone exon and/or in at least one intron and/or in the 3′UTR of said gene.

Said disease or disorder can e.g., be a trinucleotide repeat disease ordisorder, a tetranucleotide repeat disease or disorder, or apentanucleotide repeat disease or disorder.

Said disease or disorder can e.g., be any disease or disorder selectedfrom the group consisting of DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA,SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2, SCA10, SPD1, OPMD, CCD,HPE5, HFG syndrome, BPES, EIEE1, FRAXA, FXTAS and FRAXE (cf. Table 6above).

According to an aspect of the application, when said at least one DNAnucleic acid is a double-stranded nucleic acid, at least one of its twostrands (either only one of them or both of them) contains nucleotide(s)T in DNA tandem repeat unit.

Said disease or disorder can e.g., be any disease or disorder selectedfrom the group consisting of DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA,SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2 and SCA10 (cf. Table 6above).

More particularly, said disease or disorder is DM1.

Said disease and disorders are described in Table 6 above. Table 7identifies the at least one DNA nucleic acid, which is involved in eachof said diseases or disorders, respectively. Table 8 identifies thenature of the DNA tandem repeat unit that is contained in said at leastone DNA nucleic acid. Table 8 also provides the normal average range ofDNA tandem repeat units that are contained in said at least one DNAnucleic acid. A number of DNA tandem repeat units above said normalaverage range is generally considered to be an abnormal number of DNAtandem repeat units, i.e., it is then generally considered that the atleast one DNA tandem repeat is an expanded or abnormally-expanded DNAtandem repeat.

The application notably relates to the use of at least one DNA-bindingpolypeptide of the application, more particularly the use of a first andsecond DNA-binding polypeptides of the application (as above defined),or the use of at least one nucleic acid or vector of the application (asabove defined), more particularly the use of a first and second nucleicacids or vectors of the application (as above defined), wherein said useis in the manufacture of a medicament for treating and/or palliatingand/or preventing a disease or disorder involving at least one DNAtandem repeat, more particularly a disease or disorder as above defined.

The application also relates to said at least one DNA-bindingpolypeptide of the application, more particularly to said first andsecond DNA-binding polypeptides of the application, or to said at leastone nucleic acid or vector of the application, or to said first andsecond nucleic acids of the application, for its/their use as amedicament.

The application also relates to said at least one DNA-bindingpolypeptide of the application, more particularly to said first andsecond DNA-binding polypeptides of the application, or to said at leastone nucleic acid or vector of the application, or to said first andsecond nucleic acids of the application, for its/their use in thetreatment and/or palliation and/or prevention of a disease or disorderinvolving at least one DNA tandem repeat, more particularly a disease ordisorder as above defined.

The application also relates to a method for producing a drug ormedicament that is useful in the treatment and/or palliation and/orprevention of a disease or disorder involving at least one DNA tandemrepeat, more particularly a disease or disorder as above defined. Saidmethod comprises:

-   -   producing said at least one DNA-binding polypeptide of the        application, more particularly said first and second DNA-binding        polypeptides of the application, and/or producing said at least        one nucleic acid or vector of the application, more particularly        said first and second nucleic acids of the application, and/or        producing a composition or kit of the application,    -   formulating said polypeptide(s) and/or nucleic acid(s) or        vector(s) and/or composition or kit as a drug or medicament        (more particularly, mixing said polypeptide(s) and/or nucleic        acid(s) or vector(s) with at least one vehicle or carrier e.g.,        at least one vehicle or carrier as above defined).

A product of the application can induce a double-strand break in adouble-stranded DNA nucleic acid. More particularly, a product of theapplication can induce a double-strand break specifically in adouble-stranded DNA nucleic acid.

A product of the application can act by cleaving said at least one DNAtandem repeat, more particularly by reducing the number of unitscontained in said at least one DNA tandem repeat, more particularly byfully or partially deleting said at least one DNA tandem repeat.

According to an advantageous aspect of the application, a product of theapplication allows a deletion or reduction of said at least one DNAtandem repeat down to a non-abnormal number of repeat units, i.e., downto below the abnormal range (cf. e.g., Table 8 for the average normalrange of DNA tandem repeat units that is generally observed inillustrative disease or disorders).

The example below illustrates that the efficiency of a product of theapplication in achieving said deletion or reduction is very high (near100% in heterozygous and homozygous yeast cells).

The example below illustrates that a product of the application can actwithout inducing an increase in the mutation rate and without inducingany large genomic rearrangement, such as aneuploidy, segmentalduplication or translocation.

Advantageously, a means of the application is less toxic than the priorart means, more particularly than the Zinc Finger prior art means.

Advantageously, a means of the application does not induce any lengthalteration or mutation at off-target locations, e.g., innon-pathological genes, which comprise the same repeat unit as thepathological gene. It is notably the case when the DNA target site ofsaid first DNA-polypeptide is a non-overlapping DNA target site (asdefined above) and when the DNA target site of said secondDNA-polypeptide is an overlapping DNA target site (as defined above).Please see FIG. 1B for an illustration of such a configuration.

It is believed that it is the first demonstration that the shortening ofa DNA tandem repeat to lengths below pathological thresholds in humanscan be induced with 100% efficacy and a high specificity.

Reduction in size of an abnormally-expanded tandem repeat unit providesa genetic treatment and/or palliation and/or prevention of the diseaseor disorder. Indeed it has been demonstrated that, when a largetrinucleotide repeat contraction of an expanded myotonic dystrophyallele occurred during transmission from father to daughter, completeclinical examination of the daughter showed no sign of myotonicdystrophy symptoms (O'Hoy et al. 1993).

Hence, a product of the application actually provides a means for genetherapy and/or palliation and/or prevention of said diseases ordisorders.

The application also relates to a method of treatment of a subject inneed thereof, which comprises administering at least one product of theapplication to said subject. Said subject can e.g., be a mammal (e.g., anon-human mammal or a human), more particularly a human.

Said at least one product can more particularly be at least oneDNA-binding polypeptide of the application, at least one polypeptide set(composition or kit) of the application, at least one nucleic acid, atleast one set of nucleic acids, at least one liposome, at least onenanoparticle, at least one vector or at least one cell of theapplication.

Said at least one product can more particularly be at least onepharmaceutical composition, medicament or drug of the application.

The application also relates to the (in vitro) use of at least oneproduct of the application in the selection of a product suitable forcleavage and/or reduction in size, and/or full or partial deletion of atleast one (expanded or abnormally-expanded) DNA tandem repeat, moreparticularly at least one (expanded or abnormally-expanded) DNA tandemrepeat in a DNA nucleic acid, more particularly at least one (expandedor abnormally-expanded) DNA tandem repeat in a double-stranded DNAnucleic acid.

The application also relates to a method for identifying a productuseful in the treatment and/or palliation and/or prevention of a diseaseor disorder as above defined, which comprises:

-   -   in vitro growing cells, which comprise at least DNA nucleic        acid, wherein said at least one DNA nucleic acid comprises said        at least one DNA tandem repeat to be cleaved and/or reduced in        size and/or fully or partially deleted, wherein said cells are        the cells of a cell line (e.g., a cell line, which is considered        by the person of average skill in the art as a model of one or        several of said diseases or disorders, e.g., one or several of        the diseases or disorders of Table 6 above), or wherein said        cells are cells, which have been collected from a mammal, more        particularly from a non-human mammal or from a human, more        particularly from a human in need of said treatment and/or        palliation and/or prevention (e.g., a human affected by at least        one disease or disorder listed in Table 6 above),    -   contacting at least one cell of said cells with at least one        product of the application,    -   contacting at least one other of said cells with at least one        other product of the application, and    -   selecting the at least one product, which achieves said cleavage        and/or reduction in size and/or full or partial deletion with        the highest efficiency and/or with the lowest undesired side        effects.

Selecting the at least one product, which achieves said cleavage and/orreduction in size and/or full or partial deletion with the lowestundesired side effects notably encompasses selecting the at least oneproduct, which achieves said cleavage and/or reduction in size and/orfull or partial deletion, and which induces the lowest level of one orseveral side effect(s) selected from the group consisting of inducedtoxicity, rate of induced mutation, induced rate of genomicrearrangement, induced rate of aneuploidy, induced rate of segmentalduplication, induced rate of translocation, rate of off-target cleavage,e.g., in non-pathological genes, which comprise the same repeat unit asthe pathological gene.

The application also relates to a method for producing a product usefulfor fully or partially deleting a DNA tandem repeat that is contained ina double stranded DNA nucleic acid, more particularly for fully orpartially deleting a DNA tandem repeat that is contained in a doublestranded DNA nucleic acid and forms a non-linear secondary structure insaid double stranded DNA nucleic acid (more particularly a secondarystructure, which is a hairpin, a triple helix or a tetraplex structure).Said double-stranded DNA nucleic acid is as above defined and can e.g.,be contained in a chromosome, more particularly a gene that is containedin a chromosome, more particularly in a human chromosome, moreparticularly a human gene that is contained in a chromosome. Said fullor partial deletion is a deletion or excision of all or several of therepeated units of said DNA tandem repeat, more particularly a specificdeletion or excision of all or several of the repeated units of said DNAtandem repeat. Said method comprises producing a pair of DNA-bindingpolypeptides of the application (i.e., a first DNA-binding polypeptideand a second DNA-binding polypeptide as defined above for a polypeptideset, or mixed set, of the application), e.g., according to the method ofthe application. Said pair of DNA-binding polypeptides is a productuseful for said full or partial DNA tandem repeat deletion.

At least one of said first and second DNA-binding polypeptides is aDNA-binding polypeptide of the application, more particularly aDNA-binding polypeptide of the application, the DNA target of which is anon-overlapping or an overlapping DNA target site as defined above, moreparticularly a DNA-binding polypeptide of the application, the DNAtarget of which is a non-overlapping DNA target site as defined above.Advantageously, said first DNA-binding polypeptide is a DNA-bindingpolypeptide of the application, the DNA target of which is anon-overlapping DNA target site as defined above, and said secondDNA-binding polypeptide is a DNA-binding polypeptide of the application,the DNA target of which is an overlapping DNA target site as definedabove.

The application also relates to a method for inducing (or generating),more particularly in vitro inducing (or generating), a double-strand DNAbreak (into a double-stranded DNA nucleic acid).

Said method comprises placing a double-stranded DNA into contact with afirst DNA-binding polypeptide and with a second DNA-binding polypeptide(said first DNA-binding polypeptide and said second DNA-bindingpolypeptide are as defined above for a polypeptide set, or mixed set, ofthe application), or with nucleic acid(s) coding for said first andsecond DNA-binding polypeptides, or with a composition or kit, whichcomprises said first DNA-binding polypeptide and said second DNA-bindingpolypeptide and/or which comprises nucleic acid(s) coding for said firstand second DNA-binding polypeptides (cf. above).

At least one of said first and second DNA-binding polypeptides is aDNA-binding polypeptide of the application, more particularly aDNA-binding polypeptide of the application, the DNA target of which is anon-overlapping or an overlapping DNA target site as defined above, moreparticularly a DNA-binding polypeptide of the application, the DNAtarget of which is a non-overlapping DNA target site as defined above.Advantageously, said first DNA-binding polypeptide is a DNA-bindingpolypeptide of the application, the DNA target of which is anon-overlapping DNA target site as defined above, and said secondDNA-binding polypeptide is a DNA-binding polypeptide of the application,the DNA target of which is an overlapping DNA target site as definedabove.

The application also relates to a method for fully or partiallydeleting, more particularly for in vitro fully or partially deleting, aDNA tandem repeat that is contained in a double stranded DNA nucleicacid (or a DNA tandem repeat in a double stranded DNA nucleic acid,which is contained in a chromosomal DNA, more particularly in a humanchromosomal DNA).

Said method comprises placing a double-stranded DNA into contact with afirst DNA-binding polypeptide and with second DNA-binding polypeptide(said first DNA-binding polypeptide and said second DNA-bindingpolypeptide are as defined above for a polypeptide set, or mixed set, ofthe application), or with nucleic acid(s) coding for said first andsecond DNA-binding polypeptides, or with a composition or kit, whichcomprises said first DNA-binding polypeptide and said second DNA-bindingpolypeptide and/or which comprises nucleic acid(s) coding for said firstand second DNA-binding polypeptides (cf. above).

At least one of said first and second DNA-binding polypeptides is aDNA-binding polypeptide of the application, more particularly aDNA-binding polypeptide of the application, the DNA target of which is anon-overlapping or an overlapping DNA target site as defined above, moreparticularly a DNA-binding polypeptide of the application, the DNAtarget of which is a non-overlapping DNA target site as defined above.Advantageously, said first DNA-binding polypeptide is a DNA-bindingpolypeptide of the application, the DNA target of which is anon-overlapping DNA target site as defined above, and said secondDNA-binding polypeptide is a DNA-binding polypeptide of the application,the DNA target of which is an overlapping DNA target site as definedabove.

Said first and second DNA-binding polypeptides produce or generate adouble-strand DNA break in said double-stranded DNA nucleic acid. Pleasesee FIGS. 1A, 1B and 3B for illustrations of double strand DNA breaks.Said double-stranded DNA nucleic acid is as above defined and can e.g.,be contained in a chromosome, more particularly a gene that is containedin a chromosome, more particularly a human gene that is contained in achromosome. Said double-stranded DNA nucleic acid can be an isolated orpurified DNA, or can be contained into a cell. When said double-strandedDNA nucleic acid is contained into a cell, said method may furthercomprise allowing said first and second DNA-binding polypeptides tocontact or reach said double-stranded DNA.

The application also relates to an (in vitro) method for fully orpartially deleting a DNA tandem repeat that is contained in a doublestranded DNA nucleic acid, more particularly in a chromosomal DNA, moreparticularly in a human chromosomal DNA. Said method comprises:

-   -   (in vitro) contacting a cell containing said double-stranded DNA        nucleic acid, more particularly said chromosomal DNA, with at        least one DNA-binding polypeptide of the application and/or    -   (in vitro) contacting and/or (in vitro) transfecting said cell        with at least one nucleic acid of the application. More        particularly, said method comprises:    -   contacting said cell with a first DNA-binding polypeptide of the        application and with a second DNA-binding polypeptide of the        application (said first and second DNA-binding polypeptides        being as above defined) and/or    -   contacting and/or transfecting said cell with a nucleic acid or        with nucleic acids coding for said first and second DNA-binding        polypeptides, more particularly with a first nucleic acid of the        application and with a second nucleic acid (as above defined).

The phrase “transfecting” is as defined above: it is intended with itsbroadest general meaning in the field of genetic engineering. It notablyencompasses any process of deliberately introducing a nucleic acid intoa cell (said process can be virus-mediated or not virus-mediated, saidcell can be eukaryotic or not eukaryotic).

Said cell can be an isolated or purified cell, or can be contained in anorgan or tissue, e.g., an organ or tissue, which has been collected froma subject, a patient, a mammal, a non-human mammal, a human. Said cell,organ or tissue can be a mammal cell, organ or tissue, for example ahuman cell, organ or tissue, or a non-human mammal cell, organ ortissue, such as rodent cell, organ or tissue, a rat cell, organ ortissue, a mouse cell, organ or tissue, a rabbit cell, organ or tissue, apig cell, organ or tissue. Whether it is human or non-human, said cellcan e.g., be a fibroblast cell, a neuronal cell, a skeletal muscle cell,a heart cell, a skin cell, a kidney cell. Whether it is human ornon-human, said organ or tissue can e.g., be a skeletal muscle or atissue or sample thereof, a heart or a tissue or sample thereof, skin ora tissue or sample thereof, kidney or a tissue or sample thereof.

In the application, unless specified otherwise or unless a contextdictates otherwise, all the terms have their ordinary meaning in therelevant field(s).

The term “comprising”, which is synonymous with “including” or“containing”, is open-ended, and does not exclude additional, un-recitedelement(s), ingredient(s) or method step(s), whereas the term“consisting of” is a closed term, which excludes any additional element,step, or ingredient which is not explicitly recited.

The term “essentially consisting of” is a partially open term, whichdoes not exclude additional, un-recited element(s), step(s), oringredient(s), as long as these additional element(s), step(s) oringredient(s) do not materially affect the basic and novel properties ofthe application.

Accordingly, the term “comprising” (or “comprise(s)”) hence includes theterm “consisting of” (“consist(s) of”), as well as the term “essentiallyconsisting of” (“essentially consist(s) of”).

In an attempt to help the reader of the present application, thedescription has been separated in various paragraphs and/or sectionsand/or embodiments and/or aspects. These separations should not beconsidered as disconnecting the substance of a paragraph and/or sectionand/or embodiment and/or aspect from the substance of another(other)paragraph(s) and/or section(s) and/or embodiment(s) and/or aspect(s). Tothe contrary, the present application encompasses all the combinationsof the various sections, paragraphs, embodiments and aspects that can becontemplated by the person of average skill in the art.

Each of the relevant disclosures of all references cited herein isspecifically incorporated by reference. The following examples areoffered by way of illustration, and not by way of limitation.

EXAMPLES Example 1

Trinucleotide repeat expansions are responsible for at least two dozenssevere neurological or developmental disorders in humans. Adouble-strand break between two short CAG/CTG trinucleotide repeats wasformerly shown to induce a high frequency of repeat contractions inyeast cells (Richard et al. 1999). We conceived that specificendonucleases called TALENs (described in Cermark et al. 2011) couldprovide us with a new and modular tool to induce a double-strand breakwithin a repeat array.

Here we show, using a dedicated genetic selection screen, that TALENinduction of a double-strand break into a CAG/CTG trinucleotide repeatin heterozygous diploid cells results in gene conversion of the repeattract with near 100% efficacy, de facto deleting the repeat tract.Induction of the same TALEN in homozygous diploid cells leads tocontractions of both repeat tracts to a final length of 3-13 triplets,with 100% efficacy.

High throughput sequencing of yeast colonies, before and after TALENinduction, shows that the TALEN does not increase mutation rate to alevel detectable in our experiments.

No other CAG/CTG triplet repeat of the yeast genome, besides the onethat was targeted, showed any length alteration or mutation.

No large genomic rearrangement such as aneuploidy, segmental duplicationor translocation was detected.

It is believed that it is the first demonstration that induction of adedicated TALEN in a eukaryotic diploid nucleus leads to shortening of aspecific tandem repeat tract to lengths below pathological thresholds inhumans, with 100% efficacy and a high specificity, effectively pavingthe way to gene therapy of diseases or disorders linked to tandem repeatexpansions.

In the present example, a TALEN designed to recognize and cut a CAG/CTGtrinucleotide repeat was assayed in a dedicated yeast experimentalsystem. The assay relies on a modified suppressor tRNA gene (SUP4) inwhich the natural intron was replaced by either a short spacer sequence(18 bp), hereafter called SUP4-opal) or a CAG/CTG trinucleotide repeat(125-180 bp, depending on repeat length, hereafter called sup4-(CAG)).The SUP4-opal allele is functional and suppresses an ade2-opal non-sensemutation that accumulates a red pigment into yeast cells, whereas thesup4-(CAG) is not functional (Richard et al. 1999, Richard et al. 2000).Diploid yeast cells carrying homozygous ade2-opal mutations are red ifonly one copy of SUP4-opal is present, but they revert to white if twocopies are present (FIG. 1A). Haploid cells of opposite mating typescontaining either SUP4-opal or sup4-(CAG), were transformed with one ofthe two TALEN arms. As a control, a TALEN arm modified to bind arecognition site split in two halves separated by 49 bp, was alsotransformed in one of the two haploid strains. The left arm of thissplit-TALEN should not be able to bind its cognate site and therefore nodouble-strand break should be induced (FIG. 1B). TALEN arms are carriedby multicopy plasmids (2 microns) and their expression is under thecontrol of the inducible GAL1-10 promoter (Giniger et al. 1985). Cellswere simultaneously plated on glucose and galactose media and colonieswere scored after 3-5 days of growth. Yeast survival to the TALENinduction was 81.4%±7.2%, slightly less than survival to the split-TALENinduction (96.4%, FIG. 2A). White colonies were scored and represent amajority of cells on both media, even though they are more frequent ongalactose (82.5% of white colonies) as compared to glucose (66.7%). Thissuggests that even in repressing conditions (glucose), the GAL1-10promoter shows some level of leakiness which is, associated to multicopyplasmids, apparently sufficient to induce TALEN expression. In supportof this observation, we noticed that when crossing two haploids strainscontaining a stable trinucleotide repeat and one of the two TALEN arms,none of the diploids obtained contained a repeat longer than 30triplets, strongly suggesting than even in repressing conditions, leakyexpression of both TALEN arms occur to a level high enough to inducerepeat contractions when both plasmids are in the same diploid cell (cf.FIG. 4).

DNA originating from red and white colonies was subsequently analyzed bySouthern blotting. Forty-nine out of 52 red colonies contain the twoalleles, only three colonies showed the complete deletion of thesup4-(CAG) allele (FIG. 2B). Conversely, 119 out of 120 white coloniesonly contain the SUP4-opal allele, whose signal intensity was twice theintensity detected in red colonies, suggesting that it corresponds to anear-complete deletion of the sup4-(CAG) allele. We took advantage of arestriction site polymorphism between SUP4-opal and sup4-(CAG) alleles,to discriminate between a perfect homozygotization and a largecontraction of the sup4-(CAG) allele. DNA extracted from red or whitediploid survivors was amplified and digested with enzymes recognizingone of the two alleles. In all ten white survivors analyzed,restrictions showed the presence of only the SUP4-opal allele (FIG. 2C).

Sequencing the same PCR products amplified from white diploid survivorsconfirmed that only one sequence was present, and not a mix of twodifferent sequences, as would be expected for an heterozygous SUP4Isup4locus. These experiments proved that gene conversion of the sup4-(CAG)allele by the SUP4-opal allele was more than 99% efficient followingTALEN expression. Comparatively, there was no difference between glucoseand galactose and no gene conversion was detected when inducing thesplit-TALEN (FIG. 2B).

In a second set of experiments, we built a diploid strain containing twosup4-(CAG) alleles of different lengths. In such a strain, it is notpossible to screen for white colonies, since both alleles are deficientin suppressing ade2-opal mutation. In this strain, survival to galactoseinduction dropped to 37.1%±18%, a FIG. 2.2 fold lower than survival ofthe SUP4-opallsup4-(CAG) heterozygote (FIG. 2A). This shows that cuttingboth chromosomes instead of one decreases viability by about a two-foldfactor. Molecular analysis showed that ca. 5% of colonies on glucose (2out of 37) showed a small expansion, whereas 59% (22 out of 37) ofcolonies exhibited a contracted or deleted allele (FIG. 2D), suggestingagain that some TALEN induction occurs in repressing conditions.

In galactose, 100% of the 153 colonies analyzed showed one single bandcorresponding in size to the near-complete deletion of the repeat tract.However, Southern blot resolution was not sufficient to determine ifboth alleles harbored repeats of the exact same length. DNA extractedfrom diploid survivors was therefore amplified and sequenced. In 23 outof 60 sequenced survivors (38%), only one sequence was present, as shownby good quality, evenly spaced peaks (FIG. 3A). In 37 out of 60survivors (62%), a mix of two DNA sequences was read after the repeattract, indicating that the two alleles carry repeat tracts of differentlengths. Using this approach, only the shortest of the two repeat tractlengths could be determined, and was found to range from three to 13triplets (with one exception, one sequence of 20 triplets was found).Given the size of both TALE recognition sites, we determined that theminimal spacing between the two TALE DNA-binding domains necessary toobtain active dimerization of the Fok I nuclease and subsequent DSBformation was 18 bp (FIG. 1B).

Homozygous survivors may result from iterative coordinated oruncoordinated breaks on both chromosomes, one (or two) allele(s) beingcut and repaired by intra-molecular mechanism, while the other allele isrepaired by gene conversion using the shortest one as a template (FIG.3B). Heterozygous survivors may result as before, from iterativecoordinated or uncoordinated breaks, that will not be repaired by geneconversion and will therefore lead to repeat tracts of differentlengths. This may be due to the presence of CAG repeats at DSB ends,which may impede one or more steps of homologous recombination,including correct processing of the break, subsequent formation of Rad51nucleo filament, or strand invasion of the homologous template (whichalso contains CAG repeats). In support of this hypothesis, distributionof repeat tract lengths among heterozygous and homozygous survivorsshows that homozygous tract lengths are shorter on the average (mean=7triplets) than heterozygous tract lengths (mean=9 triplets), thisdifference being very significant (Wilcoxon test, p-value=0.0021, FIG.3A). This suggests that gene conversion between repeat tracts may behindered when tract lengths are too long, probably inhibiting an earlystep in the recombination process. In these cases, intramolecular repairis favored, giving rise to longer repeat tracts of unequal lengths.However, we cannot totally exclude that heterozygous survivors resultfrom slippage occurring during DNA synthesis associated to geneconversion. When competition is possible between intra- andintermolecular repair mechanisms, intramolecular events might befavored, even though homologous recombination is highly efficient inyeast.

In order to determine TALEN specificity, particularly if an increase inoff-site mutations was associated with its expression, we completelyre-sequenced eight colonies growing on glucose plates and seven coloniesgrowing on galactose plates. Paired-end ILLUMINA reads were generatedand mapped to the S288C reference genome for each colony (cf. Table 2).After removal of duplicates, coverage of unique sequences washomogeneous in all 15 clones sequenced, showing no aneuploidy and nosegmental duplication. Among eight glucose colonies, eight uniqueheterozygous SNPs were detected, whereas among seven galactose coloniesfour unique heterozygous SNPs were detected (FIG. 3C). These numbers arenot significantly different from each other and are in good agreementwith predictions. Lynch et al. 2008 determined that the average basesubstitution rate per nucleotide site was 3.3×10⁻¹⁰ per cell division,in S. cerevisiae. Given that glucose and galactose colonies underwentapproximately only 30 cell divisions before DNA was extracted andsequenced, it was expected that most of the colonies did not contain anybase substitution. Nine colonies out of 15 did not contain any SNP,whereas the remaining contained between one (three colonies) and fourSNPs (one colony). Actually, the number of colonies without any SNP washigher for clones growing in galactose than for clones growing inglucose. Altogether, five transitions for seven transversions were found(ratio: 0.71), a proportion slightly higher than expected fortransitions (expected ratio: 0.61), but figures are small. Insertionsand deletions (Indels) of one base pair in non-monotonous DNA areexpected to be ten times less frequent than base substitutions. Indeed,we only found one deletion of a GC dinucleotide in an intergenic region.However, six indels were found in monotonous poly-A/T stretches, butmore importantly no mutation was detected in any of the naturallyoccurring (at least five triplet long) 88 CAG/CTG trinucleotide repeatsof the S288C genome. All indels and five out of twelve SNPs fall withinintergenic regions. Out of seven remaining SNPs in coding regions, twoare synonymous (third codon base) whereas five are non-synonymous andencode point mutations in five different genes (cf. Tables 3 and 4below).

TABLE 3 summary of mutations detected in the 15 sequenced colonies; basesubstitutions: Chro- mo- some Position ⁽¹⁾ Mutation Location Codon Aminoacid I 175371 T −> C Intergene — — III 300201 C −> A Intergene — — IV628439 C −> A RLI1 GTG −> TTG Val −> Leu IV 1298899 G −> T SYF1 GTT −>TTT Val −> Phe X 333003 A −> T ZAP1 ACT −> ACA Synonymous X 626414 T −>C ECM27 TTT −> CTT Phe −> Leu XI 142750 C −> T PIR1 CCG −> CCASynonymous XI 315846 T −> G Intergene — — XI 609033 A −> G PXL1 CAG −>CGG Gln −> Arg XII 823062 A −> C Intergene — — XIII 330662 C −> GIntergene — — XV 1075334 A −> G YOR389w AAC −> AGC Asn −> Ser

TABLE 4 summary of mutations detected in the 15 sequenced colonies;insertions/deletions: Chro- mo- Muta- some Position ⁽¹⁾ tion SequenceLocation I 6737 +A (A)₁₉ SEQ ID NO: 17 Intergene I 101282 +A (A)₂₄ SEQID NO: 18 Intergene II 809788 −T (T)₁₉ SEQ ID NO: 19 Intergene VI 106271+TT (T)₁₃ SEQ ID NO: 20 Intergene VII 95081 −GC Non monotonous IntergeneVII 413969 −GA (A)₂G(A)₁₂ SEQ ID NO: 21 Intergene XIII 918118 +T (T)₁₉SEQ ID NO: 19 Intergene ⁽¹⁾ mutation position according to GENBANKNC_001133 to NC_001148, PLN 6 DEC. 2008 yeast genome assembly.

We concluded that expression of a TALEN targeted to a specific CAG/CTGtrinucleotide repeat has no effect on other triplet repeats and has noeffect on the overall mutation rate of the yeast genome. Sincedeep-sequencing cannot reveal reciprocal translocations that could beinduced by the TALEN, as a last control experiment, a PFGE was run onthe heterozygous SUP4-opal/sup4-opal::CAG strain. DNA from two coloniesgrown on glucose and 20 colonies grown on galactose was preparedembedded in agarose plugs and loaded on a PFGE. All karyotypes werenormal, showing no evidence for aneuploidies, large segmentalduplications or translocations (FIG. 3D).

TALEN expression leads to trinucleotide repeat contractions with a 100%efficacy, giving rise to survivors containing homozygous or heterozygousshorter alleles.

Detailed Material, Methods & Results:

Plasmid pCLS9996 (marked with KANMX) and plasmid pCLS16715 (marked withLEU2), carrying the two TALEN arms were respectively transformed intoGFY40 strain (MATa ura3Δ851 leu2Δ1 his3Δ200 lys2Δ202 ade2-opalSUP4-opal; cf. Richard et al. 1999) or GFY6162-3D (MATa ura3Δ851 leu2Δ1his3Δ200 trp1Δ65 ade2-opal sup4-(CAG); cf. Richard et al. 2003). Pleasesee FIG. 1A.

Plasmid pCLS9996 has been deposited at the C.N.C.M. under the terms ofthe Budapest Treaty [C.N.C.M. deposit number: I-4804; deposit date underthe terms of the Budapest Treaty: 10 October 2013].

Plasmid pCLS16715 has also been deposited at the C.N.C.M. under theterms of the Budapest Treaty [C.N.C.M. deposit number: I-4805; depositdate under the terms of the Budapest Treaty: 10 Oct. 2013].

Plasmid pCLS9996 codes for the right-hand TALEN monomer that binds tothe DNA target site of SEQ ID NO: 10 (cf. FIG. 1B).

Plasmid pCLS16715 codes for the left-hand TALEN monomer that binds tothe DNA target site of SEQ ID NO: 4 (cf. FIG. 1B).

Haploids were crossed and diploids containing both TALEN arms wereselected on SC-Leu supplemented with G418 sulfate (200 μg/ml).

As a control, the split-TALEN left arm carried by pCLS9984 (marked withLEU2) was transformed in GFY6162-3D, crossed to GFY40 carrying the TALENright arm, and diploids were selected as before.

Repeat lengths were checked by Southern blot in several independantdiploids before galactose induction (cf. FIG. 4 and the associatedcomments below).

The TALEN is normally repressed on glucose medium, one copy of theactive SUP4 tRNA being insufficient to suppress the ade2-opal mutation,yeast cells are red (cf. Richard et al., 1999, Richard et al., 2000,Richard et al., 2003). In the presence of galactose, the TALEN isexpressed, binds CAG/CTG trinucleotide repeats and induces adouble-strand break (DSB) into the repeat tract. If a second copy of anactive SUP4 tRNA is generated during double-strand break repair, theade2-opal mutation will be suppressed and yeast cells will now be white(cf. FIG. 1A).

Sequences recognized by both TALE DNA-binding domains and by thesplit-TALE.

The length of the spacer, which is appropriate to induce a DSB wasdeduced from repeat tract lengths analyzed in surviving cells afterTALEN induction (length of 18 bp); cf. FIG. 1B).

Sequence Data for Plasmid pCLS9996 (C.N.C.M. I-4804):

The sequence of the insert carried by plasmid pCLS9996 is:

[SEQ ID NO: 1] GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCCGATCAAAAATCATCGCTTCGCTGATTAA TTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCTATGGTTGTTAATTTGATTC GTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGC TAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAA CGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTC GGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCA AAGAGAAGGTTTTTTTAGGCTAATCGACCTCGAGCAGATCCGCCAGGCGTGTATATAGCGTGG ATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGA TCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATA TTCTTTCCTTATACATTAGGTCCTTTGTAGCATAAATTACTATACTTCTATAGACACGCAAAC ACAAATACACAGCGGCCTTGCCACCATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATAA GGAGACCGCCGCTGCCAAGTTCGAGAGACAGCACATGGACAGCATCGATATCGCCGATCTACG CACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGC GCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCA ACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGA GGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGC CTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCT CAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACT GACGGGTGCCCCGCTCAACTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGG CAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGAC CCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCA GCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGC CAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCA GGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGC GCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCA GGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTT GCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGG CGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGG CTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGAC GGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGC CATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCT GTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAA GCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCC CCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCG GCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAG CCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGC CCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCT GGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGT GGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCC GGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGG CGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTT GACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGT CCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCAT CGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGA TCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCC TGCGCTGGATGCAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTC CGAGCTGGAGGAGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACAT CGAGCTGATCGAGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGA GTTCTTCATGAAGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGG CGCCATCTACACCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTC CGGCGGCTACAACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCA GACCAGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGA GTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCT GAACCACATCACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGA GATGATCAAGGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGAT CAACTTCGCGGCCGACTGATAACTCGAGCGATCCTCTAGACGAGCTCCTCGAGCCTGCAGCAG CTGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGT CTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTG ACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAA AAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGA GTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACC ACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTG TTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGC CTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA CCTGTCGTGCCAGCAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACA TGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACT GTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATG GCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTC CCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGG ATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTC ACATCACATCCGAACATAAACAACCATGCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGC GATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGC AATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAAC ATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGG AATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCA CCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAA ATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTC CTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGG TTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAA TGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATA ACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAG ACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGA AACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGA TGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTC ATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATAT TTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATG CGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCC AGTGTCGAAAACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGACCGC GGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTA ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCATCGATGAATTCCAC GGACTATAGACTATACTAGTATACTCCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGT CCTTTAACGAGGCCTTACCACTCTTTTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTG ATCTAAGATTCTATCTTCGCGATGTAGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGC AAAAGGCACTTCTACAATGGCTGCCATCATTATTATCCGATGTGACGCTGCAGCTTCTCAATG ATATTCGAATACGCTTTGAGGAGATACAGCCTAATATCCGACAAACTGTTTTACAGATTTACG ATCGTACTTGTTACCCATCATTGAATTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAG ATAGTATATTTGAACCTGTATAATAATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTA TTTCGGTTCCTGGAGAAACTATTGCATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGG TTCATTTTCTGCGTTTCCATCTTGCACTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGC TTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCT GCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTT CATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTG CATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTC TTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTAC TTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCG TTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCA CTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCC CGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGAT GATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTAT AGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTT TTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAA GTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAA AGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAG TCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCT CTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGA AAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCAC CTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATG CTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTG ATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTA TATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTG GATCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGAT GAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAG TCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATT TTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTTTATTGTACG ATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGAATTGCCGGTCC TATTTACTCGTTTTAGGACTGGTTCAGAATTCATCGATGCTCACTCAAAGGTCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC

The nucleic acid of SEQ ID NO: 1 (carried by plasmid pCLS9996) codes forthe TALEN arm that binds to the DNA target site of SEQ ID NO: 10 (cf.FIG. 1B). Hence, the nucleic acid of SEQ ID NO: 1 comprises a sequence,which codes for adjacent units of TAL effector tandem repeat thatdetermine recognition of the DNA target site of SEQ ID NO: 10, and whichcodes for an endonuclease. The endonuclease is the monomer of a dimericendonuclease, i.e., a Fold monomer. The sequence, which codes foradjacent units of TAL effector tandem repeat and for an endonuclease, ispreceded by a promoter and an enhancer, and is followed by a terminator.The nucleic acid of SEQ ID NO: 1 further comprises a sequence, whichcodes for a selection marker, i.e., the kanamycin selection marker. Thesequence, which codes for the selection marker, is preceded by apromoter and is followed by a terminator.

The nucleic acid of SEQ ID NO: 1 further comprises a replication origin,i.e., the 2-micron replication origin.

More particularly, the nucleic acid of SEQ ID NO: 1 (carried by plasmidpCLS9996) comprises:

-   -   a GAL10 enhancer at positions 36-401 [SEQ ID NO: 37];    -   a CYC1 promoter at positions 402-641[SEQ ID NO: 38];    -   a sequence coding for a TALEN arm (TALEN arm that binds to the        DNA target site of SEQ ID NO: 10) at positions 656-3484 [SEQ ID        NO: 39];    -   an ADH1 terminator at positions 3836-4155 [SEQ ID NO: 40];    -   a TEF promoter at positions 4357-4736 [SEQ ID NO: 41];    -   a sequence coding for the KANMX selection marker at positions        4740-5546 [SEQ ID NO: 42];    -   a TEF terminator at positions 5547-5759 [SEQ ID NO: 43]; and    -   the 2-micron replication origin at positions 6585-7929 [SEQ ID        NO: 44].

Hence, the sequences of SEQ ID NOs: 37-44 are:

(GAL10 enhancer) SEQ ID NO: 37GATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCTATGGTTGTTAATTTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAA GGTTTTTTTAGGCTAA(CYC1 promoter) SEQ ID NO: 38TCGACCTCGAGCAGATCCGCCAGGCGTGTATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGTCCTTTGTAGCATAAATTACTATACTTCTATAGACACGCAAACACAAATACACA(coding for the TALEN arm that recognizes the DNAtarget site of SEQ ID NO: 10) SEQ ID NO: 39ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATAAGGAGACCGCCGCGCCAAGTTCGAGAGACAGCACATGGACAGCATCGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACACCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACAACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCGCGGCCGACTGA (ADH1 terminator) SEQ ID NO: 40TATTGACCACACCTCTACCGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCG CAGCTCAGGGGCATGATGTG(TEF promoter) SEQ ID NO: 41TGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCC(coding for the KANMX selection marker) SEQ ID NO: 42GGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGCCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCG ACCGCGG(TEF terminator) SEQ ID NO: 43ATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCATCGATGAATTCCACGG ACTATAGACTATACTAGTATACTC(2-Micron replication origin) SEQ ID NO: 44GCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGAGAAACTAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTGGCCACGGCAGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGGCCCTTCATTGAAAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATTTTTTATAGCAAAGATTGAATAAGGCGCATTTTTCTTCAAAGCTTTATTGTACGATCTGACTAAGTTATCTTTTAATAATTGGTATTCCTGTTTATTGCTTGAAGAAT

In plasmid pCLS9996, the sequence coding for the TALEN arm (SEQ ID NO:39) comprises:

-   -   a sequence coding for 15 adjacent units of TAL effector tandem        repeat, and    -   a sequence coding for an endonuclease.

The 15 adjacent units of TAL effector tandem repeat are a N- toC-ordered series of 15 adjacent units each consisting of 34 amino acids.The last C-terminal unit of 34 amino acids is followed by one(truncated) unit of 20 amino acids.

The ordered series of 15 adjacent units determines the recognition of aspecific DNA target site (of 15 nucleotides, i.e., of SEQ ID NO: 10),whereas the (truncated) unit of 20 amino acids is not involved in thespecific recognition of said DNA target site.

The sequence coding for said 15 adjacent units of 34 amino acids is atpositions 499-2028 within the TALEN coding sequence of SEQ ID NO: 39,i.e., is:

[SEQ ID NO: 45] TTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGC.

The sequence of one of said 15 adjacent units of 34 amino acids (codingsequence comprised in SEQ ID NO: 45) is:

[SEQ ID NO: 46] LTPQQVVAIASXXGGKQALETVQRLLPVLCQAHG,  or [SEQ ID NO: 25]LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG,wherein XX is the RVD of the unit.

The N- to C-ordered series of RVDs formed by the RVDs respectivelycontained in the 15 adjacent units of TAL effector tandem repeat is:

NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD; NG; NN; HD; NG.

The N- to C-ordered series of RVDs determines the recognition of the DNAtarget site of SEQ ID NO: 10, i.e., GCTGCTGCTGCTGCT (cf. Table 5 above;cf. FIG. 1B).

The sequence coding for said truncated unit of 20 amino acids is(positions 2029-2088 within the TALEN coding sequence of SEQ ID NO: 39):

[SEQ ID NO: 47] TTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAG.

The sequence of said unit of 20 amino acids is:

[SEQ ID NO: 48] LTPQQVVAIASNGGGRPALE.

The sequence coding for the FokI monomer is at positions 2885-3481within the TALEN coding sequence of SEQ ID NO: 39, i.e., is:

[SEQ ID NO: 3] cagctggtgaagtccgagctggaggagaagaaatccgagttgaggcacaagctgaagtacgtgccccacgagtacatcgagctgatcgagatcgcccggaacagcacccaggaccgtatcctggagatgaaggtgatggagttcttcatgaaggtgtacggctacaggggcaagcacctgggcggctccaggaagcccgacggcgccatctacaccgtgggctcccccatcgactacggcgtgatcgtggacaccaaggcctactccggeggctacaacctgcccatcggccaggccgacgaaatgcagaggtacgtggaggagaaccagaccaggaacaagcacatcaaccccaacgagtggtggaaggtgtaccectccagcgtgaccgagttcaagttcctgttcgtgtccggccacttcaagggcaactacaaggcccagctgaccaggctgaaccacatcaccaactgcaacggcgccgtgctgtccgtggaggagctcctgatcggcggcgagatgatcaaggccggcaccctgaccctggaggaggtgaggaggaagttcaacaacggcgagatcaacttc gcggccgac.

The Fold monomer sequence (coded by the sequence of SEQ ID NO: 3) is:

[SEQ ID NO: 49] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD.Sequence Data for Plasmid pCLS16715 (C.N.C.M. I-4805):

The sequence of the insert carried by plasmid pCLS16715 is:

[SEQ ID NO: 2] GGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCCGATCAAAAATCATCGCTTCGC TGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCTATGGTTGTTAAT TTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCA TAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTT TCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCA CCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAA AGTTCCAAAGAGAAGGTTTTTTTAGGCTAATCGACCTCGAGCAGATCCGCCAGGCGTGTATAT AGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGA CACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCT CTAAATATTCTTTCCTTATACATTAGGTCCTTTGTAGCATAAATTACTATACTTCTATAGACA CGCAAACACAAATACACAGCGGCCTTGCCACCATGGGCGATCCTAAAAAGAAACGTAAGGTCA TCGATTACCCATACGATGTTCCAGATTACGCTATCGATATCGCCGATCTACGCACGCTCGGCT ACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACG AGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCAACACCCGGCAG CGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACG AAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGG TGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAA AACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCC CGCTCAACTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGC TGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGG TGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGC CGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATG GTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCT TGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGG TGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCA TCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGT GCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGC AGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGG AGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGC TGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCC ACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCC ACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGG AGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGG TGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGG TGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCG GCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGA CCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGC AGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCG CCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCC AGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGG CGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGC AGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGT TGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATG GCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGG CCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGATG CAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGG AGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCG AGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGA AGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACA CCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACA ACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACA AGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCC TGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCA CCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGG CCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCGCGG CCGACTGATAACTCGAGCGATCCTCTAGACGAGCTCCTCGAGCCTGCAGCAGCTGAAGCTTTG GACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCA GAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAA TAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTA TACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTC CTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACC GGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCA CAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGA GCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC AGCAGATCTATTACATTATGGGTGGTATGTTGGAATAAAAATCAACTATCATCTACTAACTAG TATTTACGTTACTAGTATATTATCATATACGGTGTTAGAAGATGACGCAAATGATGAGAAATA GTCATCTAAATTAGTGGAAGCTGAAACGCAAGGATTGATAATGTAATAGGATCAATGAATATT AACATATAAAATGATGATAATAATATTTATAGAATTGTGTAGAATTGCAGATTCCCTTTTATG GATTCCTAAATCCTCGAGGAGAACTTCTAGTATATCTACATACCTAATATTATTGCCTTATTA AAAATGGAATCCCAACAATTACATCAAAATCCACATTCTCTTCAAAATCAATTGTCCTGTACT TCCTTGTTCATGTGTGTTCAAAAACGTTATATTTATAGGATAATTATACTCTATTTCTCAACA AGTAATTGGTTGTTTGGCCGAGCGGTCTAAGGCGCCTGATTCAAGAAATATCTTGACCGCAGT TAACTGTGGGAATACTCAGGTATCGTAAGATGCAAGAGTTCGAATCTCTTAGCAACCATTATT TTTTTCCTCAACATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTG GAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGCATATACCTTTTTCAACTGAAAAATTGGG AGAAAAAGGAAAGGTGAGAGCCGCGGAACCGGCTTTTCATATAGAATAGAGAAGCGTTCATGA CTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGGACCTATTGTTTTTTCC AATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATA TATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTCGTTTTGCC AGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGA TGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGATGC TACAGGTGTCCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGTTGATGCCGTTTTGTT AGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTAAA AATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCT TTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGA ATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGA TAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCT ACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAG ATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACA TCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTAT AATCACCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTT GGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTA CGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTAT CTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGA AGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAG TACCACGGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAAAAGATTC TCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAA ATTACAAAATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTAT CAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAA CGTGATAAGGAAAAAGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACACAA AAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATTTTC TCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTT AAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTG TCAGAAACGGCCTTAACGACGTAGTCGACCTCCTCTTCAGTACTAAATCTACCAATACCAAAT CTGATGGAAGAATGGGCTAATGCATCATCCTTACCCAGCGCATGTAAAACATAAGAAGGTTCT AGGGAAGCAGATGTACAGGCTGAACCCGAGGATAATGCGATATCCCTTAGTGCCATCAATAAA GATTCTCCTTCCACGTAGGCGAAAGAAACGTTAACACACCCTGGATAACGATGATCTGGAGAT CCGTTCAACGTGGTATGTTCAGCGGATAATAGACCTTTGACTAATTTATCGGATAGTCTTTTG ATGTGAGCTTGGTCGTTGTCAAATTCTTTCTTCATCAATCTCGCAGCTTCACCAAATCCCGCT ACCAATGGGGGGGCCAAAGTACCAGATCTGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCATCGATGAATTCCACGGACTATAGACTATACTAGTATACT CCGTCTACTGTACGATACACTTCCGCTCAGGTCCTTGTCCTTTAACGAGGCCTTACCACTCTT TTGTTACTCTATTGATCCAGCTCAGCAAAGGCAGTGTGATCTAAGATTCTATCTTCGCGATGT AGTAAAACTAGCTAGACCGAGAAAGAGACTAGAAATGCAAAAGGCACTTCTACAATGGCTGCC ATCATTATTATCCGATGTGACGCTGCAGCTTCTCAATGATATTCGAATACGCTTTGAGGAGAT ACAGCCTAATATCCGACAAACTGTTTTACAGATTTACGATCGTACTTGTTACCCATCATTGAA TTTTGAACATCCGAACCTGGGAGTTTTCCCTGAAACAGATAGTATATTTGAACCTGTATAATA ATATATAGTCTAGCGCTTTACGGAAGACAATGTATGTATTTCGGTTCCTGGAGAAACTATTGC ATCTATTGCATAGGTAATCTTGCACGTCGCATCCCCGGTTCATTTTCTGCGTTTCCATCTTGC ACTTCAATAGCATATCTTTGTTAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAA CGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAA CGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAAC GCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAAC GCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCC CGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTAT AATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTG GTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGC GAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGA TTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTAT GAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTG TTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAG ATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGG TAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGT GGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAA AGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGA ATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAAC GCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATAT ATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCT ATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTA TCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATT AGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATATGCATAGTACCGAGAAAC TAGTGCGAAGTAGTGATCAGGTATTGCTGTTATCTGATGAGTATACGTTGTCCTGGCCACGGC AGAAGCACGCTTATCGCTCCAATTTCCCACAACATTAGTCAACTCCGTTAGGCCCTTCATTGA AAGAAATGAGGTCATCAAATGTCTTCCAATGTGAGATTTTGGGCCATTTTTTATAGCAAAGAT TGAATAAGGCGCATTTTTCTTCAAAGCTTTATTGTACGATCTGACTAAGTTATCTTTTAATAA TTGGTATTCCTGTTTATTGCTTGAAGAATTGCCGGTCCTATTTACTCGTTTTAGGACTGGTTC AGAATTCATCGATGCTCACTCAAAGGTCGGTAATACGGTTATCCACAGAATCAGGGGATAACG CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA CTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCA CCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTT GGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTG GCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAG CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGC AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTA TTTAGAAAAATAAACAAATAG

The nucleic acid of SEQ ID NO: 2 (carried by plasmid pCLS16715) codesfor the TALEN arm that binds to the DNA target site of SEQ ID NO: 4 (cf.FIG. 1B). Hence, the nucleic acid of SEQ ID NO: 2 comprises a sequence,which codes for adjacent units of TAL effector tandem repeat thatdetermine recognition of the DNA target site of SEQ ID NO: 4, and whichcodes for an endonuclease. The endonuclease is the monomer of a dimericendonuclease, i.e., a Fold monomer. The sequence, which codes foradjacent units of TAL effector tandem repeat and for an endonuclease, ispreceded by a promoter and an enhancer, and is followed by a terminator.

The nucleic acid of SEQ ID NO: 2 further comprises a sequence, whichcodes for a selection marker, i.e., a leucine selection marker.

The nucleic acid of SEQ ID NO: 2 further comprises a replication origin,i.e., the 2-micron replication origin.

More particularly, the nucleic acid of SEQ ID NO: 2 (carried by plasmidpCLS16715) comprises:

-   -   a GAL10 enhancer at positions 43-408 [SEQ ID NO: 37, as in        plasmid pCLS9996];    -   a CYC1 promoter at positions 409-648 [SEQ ID NO: 38, as in        plasmid pCLS9996];    -   a sequence coding for a TALEN arm (TALEN arm that binds to the        DNA target site of SEQ ID NO: 4) at positions 663-3476 [SEQ ID        NO: 50];    -   an ADH1 terminator at positions 3525-3844 [SEQ ID NO: 51];    -   a sequence coding for the LEU2 selection marker at positions        4946-6040 [SEQ ID NO: 52];    -   the 2-micron replication origin at positions 7583-8927[SEQ ID        NO: 53].

The sequences of SEQ ID NO: 37 (GAL10 enhancer) and of SEQ ID NO: 38(CYC1 promoter) are described above.

The sequences of SEQ ID NOs: 50-53 are:

(coding for the TALEN arm that binds to the DNAtarget site of SEQ ID NO: 4) SEQ ID NO: 50ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATTACCCATACGATGTTCCAGATTACGCTATCGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACACCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACAACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCG CGGCCGACTGA(ADH1 terminator) SEQ ID NO: 51TTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCT TATTGACCACACCTCTACCG(coding for the LEU2 selection marker) SEQ ID NO: 52ATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGATGCTACAGGTGTCCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGTTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACATCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCACGGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAA(2-Micron replication origin) SEQ ID NO: 53AACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCAT

In plasmid pCLS16715, the sequence coding for the TALEN arm (SEQ ID NO:50) comprises:

-   -   a sequence coding for 15 adjacent units of TAL effector tandem        repeat, and    -   a sequence coding for an endonuclease.

The 15 adjacent units of TAL effector tandem repeat are a N- toC-ordered series of 15 adjacent units each consisting of 34 amino acids.The last C-terminal unit of 34 amino acids is followed by one(truncated) unit of 20 amino acids.

The ordered series of 15 adjacent units determines the recognition of aspecific DNA target site (of 15 nucleotides, i.e., of SEQ ID NO: 4),whereas the (truncated) unit of 20 amino acids is not involved in thespecific recognition of said DNA target site.

The sequence coding for said 15 adjacent units of 34 amino acids is atpositions 481-2010 within the TALEN coding sequence of SEQ ID NO: 50,i.e., is:

[SEQ ID NO: 54] TTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACG GTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCC ATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTG TGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAG CAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCG GAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCG CTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGC AATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCC CACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTG GAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTG GTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCG GTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGC GGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTG ACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTC CAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATC GCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGC CAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAG GCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAG CAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTG TTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAAT AATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCAC GGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAG ACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTG GCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTG CTGTGCCAGGCCCACGGC.

The sequence of one of said 15 adjacent units of 34 amino acids (codingsequence comprised in SEQ ID NO: 54) is:

[SEQ ID NO: 46] LTPQQVVAIASXXGGKQALETVQRLLPVLCQAHG,  or [SEQ ID NO: 55]LTPEQVVAIASXXGGKQALETVQALLPVLCQAHG,  or [SEQ ID NO: 25]LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG,orwherein XX is the RVD of the unit.

The N- to C-ordered series of RVDs formed by the RVDs respectivelycontained in the 15 adjacent units of TAL effector tandem repeat is:

NN; NG; NN; NI; NG; HD; HD; HD; HD; HD; HD; NI; NN; HD; NI.

The N- to C-ordered series of RVDs determines the recognition of the DNAtarget site of SEQ ID NO: 4, i.e., GTGATCCCCCCAGCA (cf. Table 5 above;cf. FIG. 1B).

The sequence coding for said truncated unit of 20 amino acids is thesequence of SEQ ID NO: 47 (coding for the unit of SEQ ID NO: 48; samecoding and amino acid sequences as in plasmid pCLS9996), and is atpositions 2011-2070 within the TALEN coding sequence of SEQ ID NO: 50.

The sequence coding for the Fold monomer (same FokI monomer as in theplasmid pCLS9996) is the sequence of SEQ ID NO: 3 (coding for the Foldmonomer of SEQ ID NO: 49), and is at positions 2212-2808 within theTALEN coding sequence of SEQ ID NO: 50.

Sequence Data for the DNA Target Sites:

5′-3′ sequence of the DNA target site of the left-hand TALE (cf. FIG.1B)=

[SEQ ID NO: 4] GTGATCCCCCCAGCA

Sequence complementary to the sequence of the DNA target site of theleft-hand TALE (5′-3)=

[SEQ ID NO: 5] TGCTGGGGGGATCAC

Portion of the DNA target site of the left-hand TALE that is thesequence of the 5′ end of the tandem repeat:

[SEQ ID NO: 6] CAGCA,

Portion of the DNA target site of the left-hand TALE that is the genesequence that is immediately adjacent to the 5′ end of the tandem repeat(outside of the tandem repeat sequence):

[SEQ ID NO: 7] GTGATCCCCC

5′-3′ sequence of the spacer (cf. FIG. 1B)=

[SEQ ID NO: 8] GCAGCAGCAGCAGCAGCAGC

Sequence of the spacer (5′-3′) on the complementary strand (5′-3′)=

[SEQ ID NO: 9] GCTGCTGCTGCTGCTGCTGC

5′-3′ sequence of the DNA target site of the right-hand TALE (cf. FIG.1B)=

[SEQ ID NO: 10] GCTGCTGCTGCTGCT

Sequence complementary to the DNA target site of the right-hand TALE(5′-3′)=

[SEQ ID NO: 11] AGCAGCAGCAGCAGC

Sequence of the split left TALE DNA-binding domain (5′-3′)=

[SEQ ID NO: 12] TCGCTG-CAGGTCGGCCTCAGCCTGGCCGAAAGAAAGAAATGGTCTGTGATCCCCC- CAGCAGCAGC

Sequence complementary to the split left TALE DNA-binding domain(5′-3′)=

[SEQ ID NO: 13] GCTGCTGCTG-GTCCAGCCGGAGTCGGACCGGCTTTCTTTCTTTACCAGACACTAGGGGG- CAGCGA

Molecular analysis of survivors after TALEN induction. Please see FIGS.2A-AD.

FIG. 2A: Survival after galactose induction. Cells were grown inYPLactate for 5 hours (one generation), then plated on SC-Leu platessupplemented with 200 μg/mL G418 sulfate, containing either 20 g/Lglucose or galactose. Survival was determined as the ratio of CFU ongalactose plates over CFU on glucose plates, after 3-5 days of growth at30° C.

FIG. 2B: Molecular analysis of heterozygous diploids (SUP4-opal Isup4-(CAG))[(CAG)₁₃=SEQ ID NO: 27]. Red and white colonies were picked,total genomic DNA was extracted, digested with Eco RV, loaded on a 1%agarose gel and run overnight at 1 V/cm. The gel was vacuum transferredto a HYBOND-XL® nylon membrane (AMERSHAM) and hybridized with arandomly-labeled probe specific of a unique region downstream of SUP4.After washing, the membrane was overnight exposed on a FUJIFILMFLA-9000.

FIG. 2C: DNA extracted from survivors was PCR amplified using primerssu3/su9 and in vitro digested using restriction enzyme I-Sce I (I) orPst I (P). For each clone, numbered 1 to 20, the two lanes show theresult of restriction with one of the two enzymes. When both alleles arepresent, bands of slightly different sizes corresponding to uncutalleles are visible in both lanes (arrow labeled “Uncut”), along withrestriction products of cut alleles (arrows labeled “Cut”). When onlythe SUP4-opal allele is present, no cut product is detected in the ‘I’lane (clones 8 and 11 to 20). Note that these 20 survivors correspond tothe same clones as in FIG. 2B.

FIG. 2D: Molecular analysis of homozygous diploids(sup4-(CAG)/sup4-(CAG)). Same as FIG. 2B, except that total genomic DNAwas digested with Ssp I.

[(CAG)₂₉=SEQ ID NO: 28; (CAG)₁₅=SEQ ID NO: 29; (CAG)₃=SEQ ID NO: 30]

Karyotypes and sequencing of TALEN-induced yeast colonies. Please seeFIGS. 3A-3D. FIG. 3A: Sanger sequencing of survivors. PCR fragmentamplified with su3/su9 (FIG. 2C) was sequenced using a primer (su7)located ca. 210 bp upstream of the repeat tract.

Upper and lower graphs: when only one allele was present, one uniquesequence was read [upper graph, homozygous (CTG)₉/(CTG)₉ ((CTG)₉=SEQ IDNO: 14); the sequence reads:

(SEQ ID NO: 15)] TAGCCGGGAATG(CTG)₉GGGGGATCACAGACCATTTCTTTCTT.

When two alleles of different lengths were present, the sequence wasblurry and unreadable after the shortest of the two repeat tracts [lowergraph, heterozygous (CTG)₉/(CTG)_(n) ((CTG)₉

SEQ ID NO: 14); the sequence reads:

(SEQ ID NO: 16)] TAGCCGGGAATG(CTG)₉GGGGGATCACATACTTTTTTTTTCTTTCG.

The freeware 4PEAKS was used to visualize sequences.

Histogram at the bottom of FIG. 3B: length distribution of alleles inhomozygous and heterozygous survivors to TALEN induction. The valuesread as shown in Table 1 below.

TABLE 1 Final length of Number repeat tract Heterozygotes Homozygotes 30 4 4 0 4 5 1 4 6 1 4 7 10 4 8 9 16 9 4 6 10 3 4 11 2 0 12 1 0 13 5 0 201 0

Note that for heterozygous alleles only the length of the shortestrepeat can be precisely known, hence the statistical difference observedbetween the two distributions is even more important FIG. 3B: Two modelsproposing how heterozygous and homozygous repeats may be formedfollowing TALEN induction.

FIG. 3C: Deep sequencing of yeast genomes from yeast colonies isolatedon glucose or galactose plates. Each of the 15 yeast genomes wasre-sequenced to 700× coverage, on the average (see Table 2 below). Foreach colony, the number of unique SNPs and insertions/deletions isindicated.

TABLE 2 ILLUMINA sequencing data Initial Read read length after Medianlength trimming sequencing Origin Library Total reads (bp) (bp) depthGalactose GAL1  298 × 10⁶ 110 82 1601 X  GAL2 119.6 × 10⁶ 110 82 677 XGAL3 134.4 × 10⁶ 110 82 780 X GAL4 117.8 × 10⁶ 110 82 675 X GAL5 262.2 ×10⁶ 110 82 765 X GAL6 167.6 × 10⁶ 110 82 975 X GAL7 155.4 × 10⁶ 110 821779 x  Glucose GLU1  41.2 × 10⁶ 110 83 457 X GLU2  41.2 × 10⁶ 110 83457 x  GLU3   70 × 10⁶ 110 83 394 X GLU4  118 × 10⁶ 110 83 648 X GLU5  54 × 10⁶ 110 83 303 X GLU6   28 × 10⁶ 110 83 156 X GLU7   44 × 10⁶ 11083 249 X GLU8  100 × 10⁶ 110 83 588 X

Each library corresponds to one individual colony, collected on glucoseor galactose plates

(Origin), grown in non-selective rich medium, whose DNA was extractedand sonicated to an average size of 500 bp (BIORUPTOR, maximum power(H), 30″ ON/30″ OFF cycles, 9 cycles). DNA ends were subsequentlyrepaired with T4 DNA polymerase (15 units, NEBIOLABS) and KLENOW DNApolymerase (5 units, NEBIOLABS) and phosphorylated with T4 DNA kinase(50 units, NEBIOLABS). Repaired DNA was purified on two MINELUTE columns(QIAGEN) and eluted in 16 μl (32 μl final for each library). Addition ofa 3′ dATP was performed with KLENOW DNA polymerase (exo-) (15 units,NEBIOLABS) and home-made adapters containing a 4-bp unique tag used formultiplexing, were ligated with 2 μl T4 DNA ligase (NEBIOLABS, highconcentration, 2×10⁶ units/ml). DNA was size DNA fragments was recoveredin LOBIND microtubes (EPPENDORF). DNA was PCR amplified with ILLUMINAprimers PE1.0 and PE2.0 and PHUSION DNA polymerase (1 unit, THERMOSCIENTIFIC). Depending on PCR efficiency, 9, 12 or 15 PCR cycles wereperformed on each library. Twenty-four PCR reactions were pooled, foreach library, and purified on QIAGEN purification columns (two columnswere used for 24 PCR reactions).

Elution was performed in 60 μl (twice 30 μ1) and DNA was quantified on aspectrophotometer and on an agarose gel.

Two multiplexed libraries were loaded on each lane of a HISEQ 2000(ILLUMINA), and 110 bp paired-end reads were generated. Reads qualitywas evaluated by FASTQC v.0.10.1[http://www.bioinformatics.babraham.ac.uk/projects/fastqc/] and trimmedoff using the paired-end mode of TRIMMOMATIC v0.30[http://www.usadellab.org/cms/index.php?page=trimmomatic].

TRIMMED reads were mapped along S288C chromosomes reference sequence(GENBANK NC 001133 to NC 001148, PLN 6 Dec. 2008), plus the two SUP4alleles (SUP4-opal and sup4-(CAG)) using the paired-end mapping mode ofBWA v0.6.2 (Li and Durbin 2009) with default parameters. The output SAMfiles were converted and sorted to BAM files using SAMTOOLS v0.1.18 (Liet al. 2009).

The command IndelRealigner from GATK v2.2 (DePristo et al. 2011) wasused to realigne the reads. Duplicated reads were removed using theoption “MarkDuplicates” implemented in Picard v1.81[http://picard.sourceforge.net/]. Reads uniquely mapped to the referencesequence with a minimum mapping quality of 30 (PHRED-scaled) were kept.MPILEUP files were generated by SAMTOOLS without BAQ adjustments. SNPsand INDELs were called by the options “mpileup2snp” and “mpileup2indel”of Varscan2 v2.3.5 (Koboldt et al. 2012) with a minimum depth of 5 readsand a threshold of 0.3 for minimum variant allele frequency (strains arediploids). Mismatches were kept when they represented at least 20% ofthe reads supporting the variant on each strand. They were manuallyexamined and compared between all sequenced libraries forinterpretation.

FIG. 3D: Pulse-field gel electrophoresis of red and white colonies aftergalactose induction. Karyotypes are identical among all clones and donot show any large chromosomal rearrangement, neither on chromosome X(bearing SUP4) nor on any other chromosome.

FIG. 4:

Left: strains GFY6161-3C (MATa leu2Δ1 his3Δ200 lys2Δ202 ade2-opalsup4::(CAG)₃₀) and GFY6162-3D (MATa ura3Δ851 leu2Δ1 his3Δ200 trp1Δ65ade2-opal sup4::(CAG)₁₀₀) were respectively transformed with pCLS9996(KANMX marker) or pCLS16715 (LEU2 marker). Seven transformants wereanalyzed by Southern blot, for each strain, to estimate repeat lengthvariability after transformation. Transformant 4 in strain GFY6162-3Cshows extensive contractions of the repeat tract, but all othertransformants exhibit stable trinucleotide repeats after transformation.Right: Transformants GFY6162-3C/1 and GFY6162-3D/2 were crossed, anddiploids were selected on glucose SC-Leu plates supplemented with G418sulfate (200 μg/ml). Twelve independent diploids were analyzed bySouthern blot, as previously. None of the diploids contained the repeatband around 100 triplets, showing that it was contracted during or rightafter the cross, even though cells were crossed on glucose medium. Inthis particular cross, diploid #5 was selected for further inductionexperiments.

[(CTG)₁₂₂=SEQ ID NO: 31; (CTG)₇₂=SEQ ID NO: 32; (CTG)₃₂=SEQ ID NO: 33;(CTG)₂=SEQ ID NO: 34]

Example 2

Myotonic dystrophy (DM) is caused by a CTG repeat expansion in the 3′UTRof the DM protein kinase (DMPK) gene [(CTG)_(n).(CAG), repeat]. The sizeof the CTG repeat, which increases from generation to generation withsometimes very large expansions, is generally correlated with clinicalseverity and age at onset, providing a molecular basis for theanticipation phenomenon observed in DM1 families.

Transgenic mice carrying the human DMPK gene with a normal CTG repeat(i.e., 5-37 repeat units) or with an expanded CTG repeat (e.g.,200-3,000 CTG repeat units) were generated and bred as described inGantelet et al. 2007, Seznec et al. 2001, Gomes-Pereira et al. 2007,Panaite et al. 2011, Panaite et al. 2013.

Transgenic mice carrying about 20 CTG repeat units (DM20 mice) arecontrol mice, which do not show the DM1 phenotype.

Transgenic mice carrying 200-3,000 CTG repeat units develop the DM1phenotype, ranging from mild DM1 phenotype (e.g., mice, which carryabout 500 CTG repeat units) to severe DM1 phenotype (e.g., mice, whichcarry more than 1,300 CTG repeat units).

Fibroblast primary cells have been isolated from DM20 mice and micecarrying different lengths of expanded repeat (e.g., about 500 CTGrepeat units; more than 1,300 CTG repeat units), and have been culturedon a culture medium.

Human cells have been collected from healthy donors having a normal DMPKCTG repeat length, as well as from DM1 patients at different stages ofthe disease.

Plasmids coding for DNA-binding polypeptides of the application, such asthe TALEN described in example 1 above, have been transfected into themouse fibroblast primary cells or into the human cells.

Plasmids coding for DNA-binding polypeptides of the application, such asthe TALEN described in example 1 above, have been administered to themice, e.g., by intraveinous injection.

The effect of the TALEN on the repeat length has been determined bySouthern blot analysis and/or PCR, e.g., as described in Jansen et al.1994.

BIBLIOGRAPHIC REFERENCES

-   Bedell, V. M. et al. 2012. In vivo genome editing using a    high-efficiency TALEN system. Nature 491, 114-118, doi:nature11537    [pii] 10.1038/nature11537.-   Beurdeley, M. et al. 2013. Compact designer TALENs for efficient    genome engineering. Nat. Commun. 4, 1762, doi:ncomms2782 [pii]    10.1038/ncomms2782.-   Boch, J. et al. 2009. Breaking the code of DNA binding specificity    of TAL-type III effectors. Science 326, 1509-1512, doi:1178811 [pii]    10.1126/science.1178811.-   Bogdanove and Voytas. 2011. TAL effectors: Customizable proteins for    DNA targeting. Science 33, 1843-1846.-   Cade, L. et al. 2012. Highly efficient generation of heritable    zebrafish gene mutations using homo- and heterodimeric TALENs.    Nucleic Acids Res. 40, 8001-8010, doi:gks518 [pii]    10.1093/nar/gks518.-   Cermak, T. et al. 2011. Efficient design and assembly of custom    TALEN and other TAL effector-based constructs for DNA targeting.    Nucleic Acids Res. 39, e82, doi:gkr218 [pii] 10.1093/nar/gkr218.-   Chen, S. et al. 2013. A large-scale in vivo analysis reveals that    TALENs are significantly more mutagenic than ZFNs generated using    context-dependent assembly. Nucleic Acids Res. 41, 2769-2778,    doi:gks1356 [pii] 10.1093/nar/gks1356.-   Christian, M. et al. 2010. Targeting DNA double-strand breaks with    TAL effector nucleases. Genetics 186, 757-761,    doi:genetics.110.120717 [pii] 10.1534/genetics.110.120717.-   DePristo, M. A. et al. 2011. A framework for variation discovery and    genotyping using next-generation DNA sequencing data. Nat. Genet.    43, 491-498, doi:ng.806 [pii] 10.1038/ng.806.-   Durfee et al. 2008. The complete genome sequence of Escherichia coli    DH10B: insights into the biology of a laboratory workhorse. J.    Bacteriol. 190(7): 2597-2606.-   Gantelet et al. 2007. The expansion of 300 CTG repeats in myotonic    dystrophy transgenic mice does not induce sensory or motor    neuropathy. Acta Neuropathol. 114: 175-185.-   Giniger, E., Varnum, S. M. & Ptashne, M. 1985. Specific DNA binding    of GAL4, a positive regulatory protein of yeast. Cell 40, 767-774,    doi:0092-8674(85)90336-8 [pii].-   Gomes-Pereira et al. 2007. CTG trinucleotide repeat “big jumps”:    large expansions, small mice. PLoS Genet. 3: e52.-   Jansen et al. 1994. Gonosomal mosaicism in myotonic dystrophy    patients: involvement of mitotic events in (CTG)n repeat variation    and selection against extreme expansion in sperm. Am. J. Hum. Genet.    54: 575-585.-   Koboldt, D. C. et al. 2012. VarScan 2: somatic mutation and copy    number alteration discovery in cancer by exome sequencing. Genome    Res. 22, 568-576, doi:gr.129684.111 [pii] 10.1101/gr.129684.111.-   Li, H. and Durbin, R. 2009. Fast and accurate short read alignment    with Burrows-Wheeler transform. Bioinformatics 25: 1754-1760,    doi:btp324 [pii] 10.1093/bioinformatics/btp324.-   Li., H. et al. 2009. The sequence alignment/map format and SAMtools.    Bioinformatics 25: 2078-2079, doi:btp352 [pii]    10.1093/bioinformatics/btp352.-   Li, T. et al. 2011. TAL nucleases (TALNs): hybrid proteins composed    of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res.    39, 359-372, doi:gkq704 [pii] 10.1093/nar/gkq704.-   Lynch, M. et al. 2008. A genome-wide view of the spectrum of    spontaneous mutations in yeast. Proc. Natl. Acad. Sci. U.S.A. 105,    9272-9277, doi:0803466105 [pii] 10.1073/pnas.0803466105.-   Moscou, M. J. & Bogdanove, A. J. 2010. A simple cipher governs DNA    recognition by TAL effectors. Science 326, 1501, doi:1178817 [pii]    10.1126/science.1178817 (2009).-   McKusick, V. A. 1998. Mendelian Inheritance in Man; A Catalog of    Human Genes and Genetic Disorders. Baltimore, Md., U.S.A., Johns    Hopkins University Press, ISBN 0-8018-5742-2.-   McMurray. Mechanisms of trinucleotide repeat instability during    human development. Nat. Rev. Genet. 11(11): 786-799.-   O'Hoy, K. L. et al. 1993. Reduction in size of the myotonic    dystrophy trinucleotide repeat mutation during transmission. Science    259, 809-812.-   Panaite et al. 2011. Peripheral neuropathy is linked to a severe    form of myotonic dystrophy in transgenic mice. J. Neuropathol. Exp.    Neurol. 70: 678-685.-   Panaite et al. 2013. Functional and histopathological identification    of the respiratory failure in a DMSXL transgenic mouse model of    myotonic dystrophy. Dis. Model Mech. 6(3): 622-631.-   Philippe S. et al. 2006. Lentiviral vectors with a defective    integrase allow efficient and sustained transgene expression in    vitro and in vivo. PNAS 103(47): 17684-17689.-   Qiu, Z. et al. 2013. High-efficiency and heritable gene targeting in    mouse by transcription activator-like effector nucleases. Nucleic    Acids Res., doi:gkt258 [pii] 10.1093/nar/gkt258. Remington: The    Science and Practice of Pharmacy”, 20^(th) edition, Mack Publishing    Co.; and “Pharmaceutical Dosage Forms and Drug Delivery Systems”,    Ansel, Popovich and Allen Jr., Lippincott Williams and Wilkins.-   Richard, G.-F., Dujon, B. & Haber, J. E. 1999. Double-strand break    repair can lead to high frequencies of deletions within short    CAG/CTG trinucleotide repeats. Mol. Gen. Genet. 261, 871-882.-   Richard, G.-F., Goellner, G. M., McMurray, C. T. &    Haber, J. E. 2000. Recombination-induced CAG trinucleotide repeat    expansions in yeast involve the MRE11/RAD50/XRS2 complex. EMBO J.    19, 2381-2390.-   Richard, G.-F., Cyncynatus, C. & Dujon, B. 2003. Contractions and    expansions of CAG/CTG trinucleotide repeats occur during ectopic    gene conversion in yeast, by a MUS81-independent mechanism. J. Mol.    Biol. 326, 769-782 (2003).-   Seznec et al. 2001. Mice transgenic for the human myotonic dystrophy    region with expanded CTG repeats display muscular and brain    abnormalities. Hum. Mol. Genet. 10: 2717-2726.-   WO 94/18313 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 95/09233 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 99/55892 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 2006/010834 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 2009/019612 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 2011/072246 and its national counterparts, including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 2010/079430 and its national counterparts, including its US    counterpart(s) (including the US continuation and divisional    applications).-   WO 2012/015938 and its national counterparts, including its US    national counterpart(s) (including the US continuation and    divisional applications).-   WO 2013/068430 and its national counterparts including its US    counterpart(s) (including the US continuation and divisional    applications).

1. A composition or kit comprising a first DNA-binding polypeptide and asecond DNA-binding polypeptide, wherein said first DNA-bindingpolypeptide is different from said second DNA-binding polypeptide,wherein each of said first and second DNA-binding polypeptides binds toa DNA nucleic acid comprising at least one DNA tandem repeat, whereinthe DNA nucleic acid to which said first DNA-binding polypeptide bindsis one strand of a double-stranded nucleic acid, wherein the DNA nucleicacid to which said second DNA-binding polypeptide binds is the otherstrand of the same double-stranded nucleic acid, wherein saiddouble-stranded DNA nucleic acid is a gene involved in a neurologicaland/or muscular and/or skeletal disorder or disease involving said atleast one DNA tandem repeat, wherein each of said first and secondDNA-binding polypeptides comprises a TAL effector tandem repeatconsisting of adjacent units of TAL effector tandem repeat, wherein theordered series of RVDs formed by the RVDs respectively contained in saidadjacent units of TAL effector tandem repeat, in N- to C-orientation, isan ordered series of amino acids, which determines the recognition ofthe 5′-3′ nucleotide sequence of a DNA target site contained in thestrand of double-stranded DNA nucleic acid to which said DNA-bindingpolypeptide binds, wherein the sequence of said DNA target site is: i. afragment of said strand of double-stranded DNA nucleic acid consistingof a fragment of said at least one DNA tandem repeat, wherein saidfragment comprises more than one copy of said DNA sequence unit of saidat least one DNA tandem repeat, or ii. a fragment of said strand ofdouble-stranded DNA nucleic acid, which starts outside the sequence ofsaid at least one DNA tandem repeat and ends within the sequence of saidat least one DNA tandem repeat, or conversely, which starts within thesequence of said at least one DNA tandem repeat and ends outside thesequence of said at least one DNA tandem repeat, wherein each of saidfirst and second DNA-binding polypeptides is directly or indirectlylinked to one endonuclease monomer or to one fragment of endonucleasemonomer, wherein said fragment of endonuclease monomer still comprisesthe catalytic domain of said endonuclease monomer, and wherein saidfirst and second DNA-binding polypeptides induce a partial or completedeletion of said at least one DNA tandem repeat.
 2. The composition orkit of claim 1, wherein the DNA target site of said first DNA-bindingpolypeptide is a DNA target site as defined in claim 1 i. and the DNAtarget site of said second DNA-binding polypeptide is a DNA target siteas defined in claim 1 ii.
 3. The composition or kit of claim 1 or 2,wherein the sequence of the DNA target site of said first DNA-bindingpolypeptide is different from the sequence of the DNA target site ofsaid second DNA-binding polypeptide.
 4. The composition or kit of anyone of claims 1-3, wherein the endonuclease monomer or endonucleasemonomer fragment of said first DNA-binding polypeptide dimerizes withthe endonuclease monomer or endonuclease monomer fragment of said secondDNA-binding polypeptide, when said first and second DNA-bindingpolypeptides are bound to their respective DNA target sites.
 5. Thecomposition or kit of any one of claims 1-4, wherein said dimericendonuclease is the endonuclease FokI.
 6. The composition or kit of anyone of claims 1-5, wherein the nucleotide spacer length between the DNAtarget site of said first DNA-binding polypeptide and the DNA targetsite of said second DNA-binding polypeptide is of 15-24 nucleotides. 7.The composition or kit of any one of claims 1-6, wherein said first andsecond DNA-binding polypeptides induce a double-strand breakspecifically in said double-stranded DNA nucleic acid.
 8. Thecomposition or kit of any one of claims 1-7, wherein each of said firstand second DNA-binding polypeptides further comprises a NuclearLocalization Signal (NLS).
 9. The composition or kit of any one ofclaims 1-8, wherein none of said first and second DNA-bindingpolypeptides comprises the acidic transcriptional Activator Domain (AD)of a TAL effector.
 10. A composition or kit, which comprises a firstnucleic acid and a second nucleic acid, wherein said first nucleic acidcodes for a first DNA-binding polypeptide, wherein said second nucleicacid codes for a second DNA-binding polypeptide, wherein said firstDNA-binding polypeptide is different from said second DNA-bindingpolypeptide, and wherein said first DNA-binding polypeptide and saidsecond DNA-binding polypeptide are as defined in any one of claims 1-9.11. A composition or kit, which comprises: a first recombinant nucleicacid vector and a second recombinant nucleic acid vector, wherein saidfirst recombinant nucleic acid vector codes for a first DNA-bindingpolypeptide, wherein said second recombinant nucleic acid vector codesfor a second DNA-binding polypeptide, wherein said first DNA-bindingpolypeptide is different from said second DNA-binding polypeptide, andwherein said first DNA-binding polypeptide and said second DNA-bindingpolypeptide are as defined in any one of claims 1-9; and/or comprising afirst lentiviral vector pseudotyped particle and a second lentiviralvector pseudotyped particle, wherein said first lentiviral vectorpseudotyped particle codes for a first DNA-binding polypeptide, whereinsaid second lentiviral vector pseudotyped particle codes for a secondDNA-binding polypeptide, wherein said first DNA-binding polypeptide isdifferent from said second DNA-binding polypeptide, and wherein saidfirst DNA-binding polypeptide and said second DNA-binding polypeptideare as defined in any one of claims 1-9.
 12. The composition or kit ofany one of claims 1-11, which is for use in the treatment and/orpalliation and/or prevention of said neurological and/or muscular and/orskeletal disorder or disease.
 13. The composition or kit for the useaccording to claim 12, wherein said first and second DNA-bindingpolypeptides induces a deletion of said at least one DNA tandem repeatto a length below the pathological threshold.
 14. The composition or kitfor the use according to claim 12 or 13, wherein said neurologicaland/or muscular and/or skeletal disease or disorder is a trinucleotide,tetranucleotide or pentanucleotide disease or disorder.
 15. Thecomposition or kit for the use according to any one of claims 12-14,wherein said DNA sequence unit of said at least one DNA tandem repeat is^(5′)CTG^(3′), ^(5′)TTG^(3′), ^(5′)GTC^(3′), ^(5′)CCTG^(3′),^(5′)ATTCT^(3′) or ^(5′)AGAAT^(3′).
 16. The composition or kit for theuse according to any one of claims 12-15, wherein the sequence of saidat least one DNA tandem repeat forms a secondary structure, which is ahairpin, a triple helix or a tetraplex secondary structure.
 17. Thecomposition or kit for the use according to any one of claims 12-16,wherein said neurological and/or muscular and/or skeletal disease ordisorder is a trinucleotide, tetranucleotide or pentanucleotide diseaseor disorder.
 18. The composition or kit for the use according to any oneof claims 12-17, wherein said neurological and/or muscular and/orskeletal disease or disorder is DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA,SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2 or SCA10.
 19. Thecomposition or kit for the use according to any one of claims 12-18,wherein said gene involved in a neurological and/or muscular and/orskeletal disorder or disease is: a gene coding for DMPK, ATXN8, PPP2R2B,JPH3, AR, HTT, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, COMP,ZNF9 or ATXN10, or the human gene coding for DMPK, ATXN8, PPP2R2B, JPH3,AR, HTT, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, COMP, ZNF9 orATXN10, and wherein said gene comprises said at least one DNA tandemrepeat.
 20. The composition or kit for the use according to any one ofclaims 12-19, wherein said adjacent units of TAL effector tandem repeatcomprise one or several copy(ies) of at least one sequence selected fromthe group consisting of SEQ ID NOs: 25, 26, 46 and 55, and/or compriseone or several copy(ies) of at least one of the sequences of TALeffector tandem repeat units of the DNA-binding polypeptide, which iscoded by the plasmid deposited at the Collection Nationale de Culture deMicroorganismes (C.N.C.M.), Paris, France, under deposit number I-4804or under deposit number I-4805.
 21. The composition or kit for the useaccording to any one of claims 12-20, wherein said DNA target site ofclaim 1 i. is the sequence of SEQ ID NO: 10 or 11, and/or wherein saidDNA target site of claim 1 ii. is the sequence of SEQ ID NO: 4 or 5,and/or wherein said first and second DNA-binding polypeptides are thepolypeptides coded by the sequences of SEQ ID NOs: 1 and 2,respectively.
 22. A method for producing a product that is useful forfully or partially deleting a DNA tandem repeat that is contained in adouble stranded DNA, wherein said DNA tandem repeat forms, in saiddouble stranded DNA, a secondary structure, which is a hairpin, a triplehelix or a tetraplex structure, wherein said method comprises producinga pair of DNA-binding polypeptides, wherein said pair of DNA-bindingpolypeptides is a first DNA-binding polypeptide and a second DNA-bindingpolypeptide as defined in any one of claims 1-9, and wherein said pairof DNA-binding polypeptides is a product useful for said full or partialDNA tandem repeat deletion.
 23. A method for fully or partially deletingin vitro a DNA tandem repeat that is contained in a double stranded DNA,wherein said method comprises placing in vitro said double-stranded DNAinto contact with: a first DNA-binding polypeptide and a secondDNA-binding polypeptide as defined in any one of claims 1-9, whereinsaid first and second DNA-binding polypeptides induce the full orpartial deletion of said DNA tandem repeat, or with a first nucleic acidand a second nucleic acid as defined in claim 10, wherein the first andsecond DNA-binding polypeptides, which are coded by said first andsecond nucleic acids induce the full or partial deletion of said DNAtandem repeat, or with a first recombinant nucleic acid vector and asecond recombinant nucleic acid vector as defined in claim 11, whereinthe first and second DNA-binding polypeptides, which are coded by saidfirst and second recombinant nucleic acid vectors, induce the full orpartial deletion of said DNA tandem repeat, or with a first lentiviralvector pseudotyped particle and a second lentiviral vector pseudotypedparticle as defined in claim 11, wherein the first and secondDNA-binding polypeptides, which are coded by said first and secondlentiviral vector pseudotyped particles induce the full or partialdeletion of said DNA tandem repeat.